Reducing levels of nicotinic alkaloids in plants

ABSTRACT

Two genes, A622 and NBB1, can be influenced to achieve a decrease of nicotinic alkaloid levels in plants. In particular, suppression of one or both of A622 and NBB1 may be used to decrease nicotine in tobacco plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/685,361, filed Apr. 13, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/082,953, filed Apr. 8, 2011, now U.S. Pat. No.9,029,656, which is a continuation of U.S. patent application Ser. No.11/579,661, filed Nov. 6, 2006, now U.S. Pat. No. 8,791,329, which isthe National Phase of International Patent Application No.PCT/IB2006/001741, filed Feb. 28, 2006, which claims priority from U.S.Provisional Patent Application No. 60/656,536, filed Feb. 28, 2005. Thecontents of these applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology and thedown-regulation of alkaloid synthesis. More specifically, the inventionrelates to methodology and constructs for reducing nicotinic alkaloidsin a plant, particularly but not exclusively in a tobacco plant.

BACKGROUND OF THE INVENTION

Presently, several methods exist for reducing nicotinic alkaloids, suchas nicotine, in plants. A low-nicotine strain of tobacco has beenemployed, for instance, as breeding stock for low-nicotine cultivars.Legg et al., Crop Sci 10:212 (1970). Genetic engineering methods alsocan be used to reduce nicotine levels. For example, U.S. Pat. No.5,369,023, and No. 5,260,205 discuss decreasing nicotine levels viaantisense targeting of an endogenous putrescine methyl transferase (PMT)sequence. Voelckel et al., Chemoecology 11:121-126 (2001). The tobaccoquinolate phosphoribosyl transferase (QPT) gene has been cloned,Sinclair et al., Plant Mol. Biol. 44: 603-617 (2000), and its antisensesuppression provided significant nicotine reductions in transgenictobacco plants. Xie et al., Recent Advances in Tobacco Science 30: 17-37(2004). See also U.S. Pat. Nos. 6,586,661 and 6,423,520.

Several nicotine biosynthesis enzymes are known. For instance, seeHashimoto et al., Plant Mol. Biol. 37:25-37 (1998); Reichers & Timko,Plant Mol. Biol. 41:387-401 (1999); Imanishi et al., Plant Mol. Biol.38:1101-1111 (1998). Still, there is a continuing need for additionalgenetic engineering methods for further reducing nicotinic alkaloids.When only PMT is down-regulated in tobacco, for example, nicotine isreduced but anatabine increases by about 2-to-6-fold. Chintapakorn &Hamill, Plant Mol. Biol. 53: 87-105 (2003); Steppuhn, et al., PLoS Biol2(8): e217: 1074-1080 (2004). When only QPT is down-regulated, a fairamount of alkaloids remain. See U.S. Plant Variety Certificate No.200100039.

Reducing total alkaloid content in tobacco would increase the value oftobacco as a biomass resource. When grown under conditions that maximizebiomass, such as high density and multiple cuttings, tobacco can yieldmore than 8 tons dry weight per acre, which is comparable with othercrops used for biomass. Large-scale growing and processing ofconventional tobacco biomass has several drawbacks, however. Forexample, significant time and energy is spent extracting, isolating, anddisposing tobacco alkaloids because conventional tobacco biomass,depending on the variety, contains about 1 to about 5 percent alkaloids.On a per acre basis, conventional tobacco biomass contains approximatelyas much as 800 pounds of alkaloids. Also, people handling tobacco maysuffer from overexposure to nicotine, commonly referred to as “greentobacco disease.”

Reduced-alkaloid tobacco is more amenable for non-traditional purposes,such as biomass and derived products. For example, it is advantageous touse reduced-alkaloid tobacco for producing ethanol and proteinco-products. U.S. published application No. 2002/0197688. Additionally,alkaloid-free tobacco or fractions thereof may be used as a forage crop,animal feed, or a human nutritive source. Id.

Beyond these benefits associated with reducing nicotine, more successfulmethods are needed to assist smokers in quitting smoking. Nicotinereplacement therapy (NRT) is not very effective as a smoking cessationtreatment because its success rate is less than 20 percent after 6 to12-months from the end of the nicotine replacement period. Bohadana etal., Arch Intern. Med. 160:3128-3134 (2000); Croghan et al., NicotineTobacco Res. 5:181-187 (2003); Stapleton et al., Addiction 90:31-42(1995). Nicotine-reduced or nicotine-free tobacco cigarettes haveassisted smokers in quitting smoking successfully, by weaning the smokerfrom nicotine yet allowing the smoker to perform the smoking ritual.Additionally, denicotinized cigarettes relieve craving and other smokingwithdrawal symptoms. See Rose, Psychopharmacology 184: 274-285 (2006)and Rose et al., Nicotine Tobacco Res. 8:89-101 (2006).

Accordingly, there is a continuing need to identify additional geneswhose expression can be affected to decrease nicotinic alkaloid content.

SUMMARY OF THE INVENTION

Two genes, A622 and NBB1, can be influenced to achieve a decrease ofnicotinic alkaloid levels in plants. In particular, suppression of oneor both of A622 and NBB1 may be used to decrease nicotine in tobaccoplants.

Accordingly, in one aspect, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence selected from (a) anucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequencethat encodes a polypeptide having the amino acid sequence set forth inSEQ ID NO: 4; and (c) a nucleotide sequence that differs from thenucleotide sequences of (a) or (b) due to degeneracy of the genetic codeand encodes a polypeptide with NBB1 expression.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence selected from (a) a nucleotidesequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence thatencodes a polypeptide having the amino acid sequence set forth in SEQ IDNO: 2; and (c) a nucleotide sequence that differs from the nucleotidesequences of (a) or (b) due to degeneracy of the genetic code andencodes a polypeptide with A622 expression, wherein said nucleotidesequence is operatively linked to a heterologous promoter.

In another aspect, the invention provides a method for reducing analkaloid in a plant, comprising decreasing NBB1 and A622 expression.

In another aspect, the invention provides a transgcnic plant havingreduced A622 expression and alkaloid content, as well as a tobacco planthaving reduced NBB1 expression and alkaloid content. The inventionprovides also a genetically engineered plant having reduced nicotine andanatabine content.

In another aspect, the invention provides a reduced-nicotine tobaccoproduct made from a tobacco plant having reduced A622 or NBB1expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nicotine biosynthesis pathway. The abbreviations are:AO=aspartate oxidase, QS=quinolinate synthase, QPT=quinolinatephosphoribosyl transferase, ODC=ornithine decarboxylase, PMT=putrescineN-methyltransferase, and DAO=diamine oxidase.

FIG. 2A schematically illustrates pHANNIBAL.

FIG. 2B schematically illustrates pHANNIBAL-X in which the multilinkersites have been modified.

FIG. 3 depicts the scheme for preparing a plant RNAi binary vector usingthe modified pHANNIBAL-X as an intermediate plasmid.

FIG. 4 depicts the T-DNA region of pRNAi-A622.

FIG. 5 depicts nicotinic alkaloid accumulation in BY-2 cells,A662-silenced BY-2 cells, and NBB1-silenced BY-2 cells. Abbreviations:WT—wild type, nontransformed cells; vector—cells transformed with markeronly; A3, A21, A33, A43-cells transformed with a construct forsuppression of A622; N37, N40-cells transformed with a construct forsuppression of NBB1.

FIG. 6 depicts expression of A622, NBB1, and genes for known enzymes inthe nicotine biosynthesis pathway in wild-type BY-2 cells, A622-silencedBY-2 cells, and NBB1-silenced BY-2 cells. A3, A21, A33 and A43 areA622-silenced lines; N37 and N40 are NBB-silenced lines. WT is anon-transformed control. N is a negative control.

FIG. 7 depicts the T-DNA region of the inducible A622 expression vectorpXVE-A622RNAi.

FIG. 8A depicts the specific suppression of A622 in hairy root linestransformed with an inducible A622 suppression construct after inducingsuppression with estradiol. Tobacco hairy root lines were cultured with(+) or without (−) addition of estradiol into the liquid culture mediumfor 4 days, and then roots were harvested and analyzed.WT—non-transformed wild-type line; XVE-A622 RNAi #8 and XVE-A622 RNAi#10-inducible RNAi lines.

FIG. 8B illustrates reduced-nicotine content in hairy root linestransformed with an inducible A622 suppression construct after inducingsuppression with estradiol. Tobacco hairy root lines were cultured with(+) or without (−) addition of estradiol into the liquid culture mediumfor 4 days, and then roots were harvested and analyzed.WT—non-transformed wild-type line; XVE-A622 RNAi #8 and XVE-A622 RNAi#10-inducible RNAi lines.

FIG. 9 depicts RNA blot analysis of NBB1 expression and PMT expressionin root and leaf tissue of wild type tobacco and nic1, nic2, andnic1nic2 mutants.

FIG. 10 depicts an alignment of NBB1 (SEQ ID NO: 4) with Eschscholziacalifornica berberine bridge enzyme (EcBBE) (SEQ ID NO: 37).

FIG. 11 depicts a phylogenetic tree constructed using NBB1 and plantBBE-like protein sequences.

FIG. 12 depicts the T-DNA region of the NBB1 suppression vectorpHANNIBAL-NBB1 3′.

FIG. 13 depicts the reduction of nicotinic alkaloid synthesis inNBB1-suppressed tobacco hairy roots. Wild type—hairy root line producedby transformation with wild-type A. rhizogenes; vector 1 and vector2—hairy root lines produced by transformation with a vector without NBB1sequences; HN6, HN19, HN20, HN29—hairy root lines produced bytransformation with the NBB1 suppression vector pRNAi-NBB1 3′.

FIG. 14 depicts expression of NBB1, A622, and known enzymes involved innicotine biosynthesis in NBB1-silenced and control hairy root lines.

FIG. 15 depicts the T-DNA region of the NBB1 suppression vectorpANDA-NBB1full.

FIG. 16 depicts levels of nicotine in the leaves of Nicotiana tabacumplants from lines transformed with the NBB1 suppression vectorpANDA-NBB1 full.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors identified two genes, A622 and NBB1, that can beinfluenced to achieve a decrease of nicotinic alkaloid levels in plants,including but not limited to tobacco. While A622 was identifiedpreviously by Hibi et al. Plant Cell 6: 723-735 (1994), the presentinventors discovered a role for A622, heretofore unknown, in the contextof decreasing nicotine biosynthesis. The field was wholly unaware ofNBB1, before the inventor's discovery, and they also elucidated a rolefor NBB1 in an approach, according to the present invention, forreducing nicotinic alkaloid content in plants.

Accordingly, the present invention encompasses both methodology andconstructs for reducing nicotinic alkaloid content in a plant, bysuppressing A622 or NBB1 expression. That is, nicotinic alkaloid levelscan be reduced by suppressing one or both of A622 and NBB1. Pursuant tothis aspect of the invention, a plant or any part thereof is transformedwith a nucleotide sequence, expression of which suppresses at least oneof A622 and NBB1 and reduces nicotinic alkaloid content.

In another aspect of the invention, nicotine can be further suppressedin a plant by concurrently suppressing expression of any known enzyme inthe nicotine biosynthesis pathway, such as QPT or PMT, and at least oneof A622 and NBB1. In addition to decreasing nicotine, for example, thepresent invention provides a means for concurrently reducing anatabine.Thus, anatabine levels can be lowered by suppressing a nicotinebiosynthesis gene, such as QPT, and at least one of A622 and NBB1.

By means of affecting A622 and/or NBB1 expression, to the ends ofreducing nicotinic alkaloid content in a plant, numerousreduced-alkaloid plants and by-products may be obtained, in keeping withthe present invention. For example, a tobacco plant having suppressedA622 or NBB1 expression may be used for producing reduced-nicotinecigarettes, which may find use as a smoking cessation product. Likewise,reduced-nicotine tobacco may be used as a forage crop, animal feed, or asource for human nutrition.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since changes and modificationswithin the spirit and scope of the invention may become apparent tothose of skill in the art from this detailed description.

DEFINITIONS

The technical terms employed in this specification are commonly used inbiochemistry, molecular biology and agriculture; hence, they areunderstood by those skilled in the field to which this inventionbelongs. Those technical terms can be found, for example in: MOLECULARCLONING: A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook andRussel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubel et al., GreenePublishing Associates and Wiley-Interscience, New York, 1988 (withperiodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OFMETHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 5^(th) ed., vol.1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS:A LABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1997.

Methodology involving plant biology techniques are described herein andare described in detail in methodology treatises such as METHODS INPLANT MOLECULAR BIOLOGY: A LABORATORY COURSE MANUAL, ed. Maliga et al.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995.Various techniques using PCR are described, for example, in Innis etal., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press,San Diego, 1990 and in Dieffenbach and Dveksler, PCR PRIMER: ALABORATORY MANUAL, 2^(nd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2003. PCR-primer pairs can be derived from knownsequences by known techniques such as using computer programs intendedfor that purpose, e.g., Primer, Version 0.5, 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass. Methods for chemical synthesisof nucleic acids are discussed, for example, in Beaucage & Caruthers,Tetra. Letts. 22:1859-1862 (1981), and Matteucci & Caruthers, J. Am.Chem. Soc. 103:3185 (1981).

Restriction enzyme digestions, phosphorylations, ligations andtransformations were done as described in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2^(nd) ed. (1989), Cold Spring HarborLaboratory Press. All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

A622 expression is controlled by the NIC1 and NIC2 gene loci in tobaccoplants. Hibi et al., The Plant Cell, 6: 723-735 (1994). It has beenreported that A622 exhibits the same expression pattern as PMT. Shoji,T. et al., Plant Cell Physiol., 41:9:1072-1076 (2000a); Shoji, T., etal., Plant Mol Biol, 50:427-440 (2002). Both A622 and PMT are expressedspecifically in roots, particularly in the cortex and endodermis of theapical parts of the roots and root hairs. Shoji et al. (2002). Moreover,A622 and PMT have a common pattern of expression in response to NICregulation and methyl-jasmonate stimulus. A622 is induced in the rootsof Nicotiana tabacum in response to wounding of aerial tissues. Cane etal., Functional Plant Biology, 32, 305-320 (2005). In N. glauca, A622 isinduced in wounded leaves under conditions that result in QPT induction.Sinclair et al., Func. Plant Biol., 31:721-9 (2004).

The nucleic acid sequence of A662 (SEQ ID NO: 1) has been determined.Hibi et al. (1994), supra. The protein encoded by this nucleic acidsequence (SEQ ID NO: 2) resembles isoflavone reductases (IFR) andcontains an NADPH-binding motif. A622 shows homology to TP7, a tobaccophenylcoumaran benzylic ether reductase (PCBER) involved in ligninbiosynthesis. Shoji et al. (2002), supra. No PCBER activity wasobserved, however, when A622 expressed in E. coli was assayed with twodifferent substrates.

Based on co-regulation of A622 and PMT and the similarity of A622 toIFR, A622 was proposed to function as a reductase in the final steps ofnicotinic alkaloid synthesis. Hibi et al. (1994); Shoji, et al. (2000a).No IFR activity was observed, however, when the protein was expressed inbacteria (id.). The function of A622 was unknown previously, and therewas no understanding heretofore that A622 plays a role in nicotinesynthesis.

A622 expression refers to biosynthesis of a gene product encoded by SEQID NO: 1. A622 suppression refers to the reduction of A622 expression.A622 suppression has an ability to down-regulate nicotinic alkaloidcontent in a plant or a plant cell.

An alkaloid is a nitrogen-containing basic compound found in plants andproduced by secondary metabolism. A nicotinic alkaloid is nicotine or analkaloid that is structurally related to nicotine and that issynthesized from a compound produced in the nicotine biosynthesispathway. In the case of tobacco, nicotinic alkaloid content and totalalkaloid content are used synonymously.

Illustrative Nicotiana alkaloids include but are not limited tonicotine, nornicotine, anatabine, anabasine, anatalline,N-methylanatabine, N-methylanabasine, myosmine, anabaseine,N′-formylnornicotine, nicotyrine, and cotinine. Other very minoralkaloids in tobacco leaf are reported, for example, in Hecht, S. S. etal., Accounts of Chemical Research 12: 92-98 (1979); Tso, T. C.,Production, Physiology and Biochemistry of Tobacco Plant. Ideals Inc.,Beltsville, Md. (1990). The chemical structures of several alkaloids arepresented, for example, in Felpin et al., J. Org. Chem. 66: 6305-6312(2001).

Nicotine is the primary alkaloid in N. tabacum along with 50-60 percentof other species of Nicotiana. Based on alkaloid accumulation in theleaves, nornicotine, anatabine, and anabasine are the other foremostalkaloids in N. tabacum. Anatabine is usually not the primary alkaloidin any species but does accumulate to relatively higher amounts in 3species; anabasine is the primary alkaloid in four species. Nomicotineis the primary alkaloid in 30 to 40 percent of Nicotiana species.Depending on the variety, about 85 to about 95 percent of totalalkaloids in N. tabacum is nicotine. Bush, L. P., Tobacco Production,Chemistry and Technology, Coresta 285-291 (1999); Hoffmann, et al.,Journal of Toxicology and Environmental Health, 41:1-52, (1994).

In the present invention, nicotinic alkaloid content can be reduced in agenetically engineered plant by down-regulating at least one of A622 andNBB1. Additionally, a nicotinic alkaloid content can be lowered bydown-regulating a nicotine biosynthesis enzyme, such as QPT or PMT, andat least one of A622 and NBB1.

Anatabine is a nicotinic alkaloid. Previous studies have demonstratedthat PMT suppression reduces nicotine content but increases putrescineand anatabine levels. Chintapakorn & Hamill, Plant Mol. Biol. 53: 87-105(2003); Sato et al., Proc. Natl. Acad Sci. USA 98, 367-372. (2001);Steppuhn, A., et al., PLoS Biol 2(8): e217: 1074-1080 (2004). For thepurposes of the present invention, anatabine content can be lowered in agenetically engineered plant by down-regulating at least one of A622 andNBB1. Anatabine levels can be lowered further by down-regulating anicotine biosynthesis enzyme, such as QPT, and at least one of A622 andNBB1.

A BY-2 Tobacco Cell is a cell line established in 1960s by Japan TobaccoCo., Ltd. from a tobacco variety Bright Yellow-2. Since this cell linegrows very fast in tissue culture, it is easy to grow on a large scaleand is amenable for genetic manipulation. A BY-2 tobacco cell is widelyused as a model plant cell line in basic research. When cultured in astandard medium, a BY-2 tobacco cell does not produce nicotinicalkaloids. Addition of jasmonate into the culture medium inducesformation of nicotinic alkaloids.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase. Thoseskilled in the art also use “cDNA” to denote to a double stranded DNAmolecule that includes such a single-stranded DNA molecule and itscomplementary DNA strand. Typically, a primer complementary to portionsof mRNA is employed for the initiation of a reverse transcriptionprocess that yields a cDNA.

Expression refers to the biosynthesis of a gene product. In the case ofa structural gene, for example, expression involves transcription of thestructural gene into mRNA and the translation of the mRNA into one ormore polypeptides.

Gene refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full-length codingsequence or by any portion of the coding sequence. A gene may constitutean uninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. Moreover, a gene may containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes may be referredto as “variants” of the “native” gene.

Genetically engineered (GE) encompasses any methodology for introducinga nucleic acid or specific mutation into a host organism. For example, atobacco plant is genetically engineered when it is transformed with apolynucleotide sequence that suppresses expression of a gene, such asA622 or NBB1, and thereby reduces nicotine levels. In contrast, atobacco plant that is not transformed with a polynucleotide sequencethat suppresses expression of a target gene is a control plant and isreferred to as a “non-transformed” plant.

In the present context, the “genetically engineered” category includes“transgenic” plants and plant cells (see definition, infra), as well asplants and plant cells produced by means of targeted mutagenesiseffected, for example, through the use of chimeric RNA/DNAoligonucleotides, as described by Beetham et al., Proc. Nat'l. Acad.Sci. USA 96: 8774-8778 (1999) and Zhu et al., loc. cit. at 8768-8773, orso-called “recombinagenic olionucleobases,” as described in PCTapplication WO 03/013226. Likewise, a genetically engineered plant orplant cell may be produced by the introduction of a modified virus,which, in turn, causes a genetic modification in the host, with resultssimilar to those produced in a transgenic plant, as described herein.See, e.g., U.S. Pat. No. 4,407,956. Additionally, a geneticallyengineered plant or plant cell may be the product of any native approach(i.e., involving no foreign nucleotide sequences), implemented byintroducing only nucleic acid sequences derived from the host plantspecies or from a sexually compatible plant species. See, e.g., U.S.published application No. 2004/0107455.

A genomic library is a collection of clones that contains at least onecopy of essentially every DNA sequence in the genome.

The NBB1 sequence was identified by cDNA microarray prepared from aNicotiana sylvestris-derived cDNA library, pursuant to the protocol ofKatoh et al., Proc. Japan Acad., 79 (Ser. B): 151-154 (2003). NBB1 alsois controlled by the nicotine biosynthesis regulatory loci, NIC1 andNIC2. NBB1 and PMT have the same pattern of expression in tobaccoplants. That NBB1 is involved in nicotine biosynthesis is indicated bythe fact that NBB1, like PMT and A622, is under the control of the NICgenes and exhibits a similar pattern of expression.

NBB1 expression refers to biosynthesis of a gene product encoded by SEQID NO: 3. NBB1 suppression refers to the reduction of NBB1 expression.

NBB1 suppression has an ability to down-regulate nicotinic alkaloidcontent.

NIC1 and NIC2 loci are two independent genetic loci in N. tabacum,formerly designated as A and B. Mutations nic1 and nic2 reduceexpression levels of nicotine biosynthesis enzymes and nicotine content,generally the nicotine content of wild type>homozygous nic2>homozygousnic1>homoyzgous nic1 and homozygous nic2 plants. Legg & Collins, Can. J.Cyto. 13:287 (1971); Hibi et al., Plant Cell 6: 723-735 (1994); Reed &Jelesko, Plant Science 167:1123 (2004).

Nicotine is the major alkaloid in N. tabacum and some other species inthe Nicotiana genus. Other plants have nicotine-producing ability,including, for example, Duboisia, Anthocericis and Salpiglessis generain the Solanaceae, and Eclipta and Zinnia genera in the Compositae.

Plant is a term that encompasses whole plants, plant organs (e. g.leaves, stems, roots, etc.), seeds, and plant cells and progeny of thesame. Plant material includes, without limitation, seeds suspensioncultures, embryos, meristematic regions, callus tissues, leaves, rootsand shoots, gametophytes, sporophytes, pollen, and microspores. Theclass of plants which can be used in the present invention is generallyas broad as the class of higher plants amenable to transformationtechniques, including both monocotyledonous and dicotyledonous plants. Apreferred plant is a plant having nicotine-producing ability of theNicotiana, Duboisia, Anthocericis and Salpiglessis genera in theSolanaceae or the Eclipta and Zinnia genera in the Compositae. Aparticularly preferred plant is Nicotiana tabacum.

Protein refers to a polymer of amino acid residues.

Reduced-nicotine plant encompasses a genetically engineered plant thatcontains less than half, preferably less than 25%, and more preferablyless than 20% or less than 10% of the nicotine content of anon-transgenic control plant of the same type. A reduced-nicotine plantalso includes a genetically engineered plant that contains less totalalkaloids compared with a control plant.

A structural gene refers to a DNA sequence that is transcribed intomessenger RNA (mRNA) which is then translated into a sequence of aminoacids characteristic of a specific polypeptide. “Messenger RNA (mRNA)”denotes an RNA molecule that contains the coded information for theamino acid sequence of a protein.

Sequence identity or “identity” in the context of two nucleic acid orpolypeptide sequences includes reference to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified region. When percentage of sequence identity is used inreference to proteins it is recognized that residue positions which arenot identical often differ by conservative amino acid substitutions,where amino acid residues are substituted for other amino acid residueswith similar chemical properties, such as charge and hydrophobicity, andtherefore do not change the functional properties of the molecule. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Sequences which differ by such conservativesubstitutions are said to have “sequence similarity” or “similarity.”Means for making this adjustment are well-known to those of skill in theart. Typically this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, forexample, according to the algorithm of Meyers & Miller, Computer Applic.Biol. Sci. 4: 11-17 (1988), as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

Use in this description of a percentage of sequence identity denotes avalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Sequence identity has an art-recognized meaning and can be calculatedusing published techniques. See COMPUTATIONAL MOLECULAR BIOLOGY, Lesk,ed. (Oxford University Press, 1988), BIOCOMPUTING: INFORMATICS ANDGENOME PROJECTS, Smith, ed. (Academic Press, 1993), COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin & Griffin, eds., (Humana Press, 1994),SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, Von Heinje ed., Academic Press(1987), SEQUENCE ANALYSIS PRIMER, Gribskov & Devereux, eds. (MacmillanStockton Press, 1991), and Carillo & Lipton, SIAMJ. Applied Math. 48:1073 (1988). Methods commonly employed to determine identity orsimilarity between two sequences include but are not limited to thosedisclosed in GUIDE TO HUGE COMPUTERS, Bishop, ed., (Academic Press,1994) and Carillo & Lipton, supra. Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude but are not limited to the GCG program package (Devereux et al.,Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschulet al., J. Mol. Biol. 215: 403 (1990)), and FASTDB (Brutlag et al.,Comp. App. Biosci. 6: 237 (1990)).

Tobacco refers to any plant in the Nicotiana genus that producesnicotinic alkaloids. Tobacco also refers to products comprising materialproduced by a Nicotiana plant, and therefore includes, for example,cigarettes, cigars, chewing tobacco, snuff and cigarettes made from GEreduced-nicotine tobacco for use in smoking cessation. Examples ofNicotiana species include but are not limited to N. alata, N. glauca, N.longiflora, N. persica, N. rustica, N. sylvestris, and N. tabacum.

Tobacco-specific nitrosamines (TSNAs) are a class of carcinogens thatare predominantly formed in tobacco during curing, processing, andsmoking. Hoffman, D., et al., J. Natl. Cancer Inst. 58, 1841-4 (1977);Wiernik A et al., Recent Adv. Tob. Sci, (1995), 21: 39-80. TSNAs, suchas 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), andN′-nitrosoanabasine (NAB), are formed by N-nitrosation of nicotine andother minor Nicotiana alkaloids, such as nornicotine, anatabine, andanabasine.

Reducing nicotinic alkaloids reduces the level of TSNAs in tobacco andtobacco products.

Tobacco hairy roots refers to tobacco roots that have T-DNA from an Riplasmid of Agrobacterium rhizogenes integrated in the genome and grow inculture without supplementation of auxin and other pytohormones. Tobaccohairy roots produce nicotinic alkaloids as roots of a tobacco plant do.

Transgenic plant refers to a plant that comprises a nucleic acidsequence that also is present per se in another organism or species orthat is optimized, relative to host codon usage, from another organismor species.

A transgenic plant may be produced by any genetic transformationmethodology. Suitable transformation methods include, for example,Agrobacterium-mediated transformation, particle bombardment,electroporation, polyethylene glycol fusion, transposon tagging, andsite-directed mutagenesis. Identification and selection of a transgenicplant are well-known techniques, the details of which need not berepeated here.

A variant is a nucleotide or amino acid sequence that deviates from thestandard, or given, nucleotide or amino acid sequence of a particulargene or protein. The terms “isoform,” “isotype,” and “analog” also referto “variant” forms of a nucleotide or an amino acid sequence. An aminoacid sequence that is altered by the addition, removal or substitutionof one or more amino acids, or a change in nucleotide sequence, may beconsidered a “variant” sequence. The variant may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties, e.g., replacement of leucine with isoleucine. Avariant may have “nonconservative” changes, e.g., replacement of aglycine with a tryptophan. Analogous minor variations may also includeamino acid deletions or insertions, or both. Guidance in determiningwhich amino acid residues may be substituted, inserted, or deleted maybe found using computer programs well known in the art such as VectorNTI Suite (InforMax, MD) software. “Variant” may also refer to a“shuffled gene” such as those described in Maxygen-assigned patents.

The present invention is not limited to the particular methodology,protocols, vectors, and reagents, etc., described here, as these mayvary. Furthermore, this specification employs the above-discussedterminology for the purpose of describing particular embodiments onlyand not to limit the scope of the invention.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, a reference to “a gene” isa reference to one or more genes and encompasses equivalents thereofthat are known to the skilled person, and so forth.

Polynucleotide Sequences

Nicotinic alkaloid biosynthesis genes have been identified in severalplant species, exemplified by Nicotiana plants. Accordingly, the presentinvention embraces any nucleic acid, gene, polynucleotide, DNA, RNA,mRNA, or cDNA molecule that is isolated from the genome of a plantspecies that down-regulates nicotinic alkaloid biosynthesis.

For example, suppression of at least one of A622 and NBB1, may be usedto down-regulate nicotine content in a plant. Additionally, nicotinicalkaloid levels can be reduced further by suppressing expression of anicotine biosynthesis gene, such as at least one of QPT and PMT, and atleast one of A622 and NBB1. Plants with suppression of multiple genesmay be obtained by regeneration of plants from plant cells geneticallyengineered for suppression of multiple genes or by crossing a firstplant genetically engineered for suppression of a nicotine biosynthesisgene with a second plant genetically engineered for suppression of atleast one of A622 and NBB1.

In one aspect, the invention provides an isolated nucleic acid moleculecomprising SEQ ID NO: 1; polynucleotide sequences encoding a polypeptideset forth in SEQ ID NO: 2; polynucleotide sequences which hybridize toSEQ ID NO: 1 and encode an A622 polypeptide; and polynucleotidesequences which differ from SEQ ID NO: 1 due to the degeneracy of thegenetic code. A peptide encoded by SEQ ID NO: 1 is a further aspect ofthe invention and is set forth in SEQ ID NO: 2.

In another aspect, the invention provides an isolate nucleic acidmolecule comprising SEQ ID NO: 3; polynucleotide sequences encoding apolypeptide set forth in SEQ ID NO: 4; polynucleotide sequences whichhybridize to SEQ ID NO: 3 and encode an NBB1 polypeptide; andpolynucleotide sequences which differ from SEQ ID NO: 3 due to thedegeneracy of the genetic code. A peptide encoded by SEQ ID NO: 3 is afurther aspect of the invention and is set forth in SEQ ID NO: 4

The invention further provides nucleic acids that are complementary toSEQ ID NO: 1 or 3, as well as a nucleic acid, comprising at least 15contiguous bases, that hybridizes to SEQ ID NO: 1 or 3 under moderate orhigh stringency conditions, as described below. For the purposes of thisdescription, the category of nucleic acids that hybridize to SEQ ID NO:3 is exclusive of a nucleic acid having the sequence of SEQ ID NO: 559,disclosed in published international application WO 03/097790, and ofany fragment thereof.

In a further embodiment, a siRNA molecule of the invention comprises apolynucleotide sequence that suppresses expression of either of SEQ IDNO. 1 or 3, although the sequences set forth in SEQ ID NO: 1 or 3 arenot limiting. A siRNA molecule of the invention can comprise anycontiguous A622 or NBB1 sequence, e.g., about 15 to about 25 or more, orabout 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguousnucleotides. In this context, too, the category of siRNA molecules isexclusive of a molecule having the nucleotide sequence of theaforementioned SEQ ID NO: 559 in WO 03/097790, as well as any fragmentthereof.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in a DNAconstruct are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells or DNAmolecules that are purified, partially or substantially, in solution.Isolated RNA molecules include in vitro RNA transcripts of the DNAmolecules of the present invention. Isolated nucleic acid molecules,according to the present invention, further include such moleculesproduced synthetically.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAor RNA may be double-stranded or single-stranded. Single-stranded DNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also called the anti-sense strand.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.).Therefore, as is known in the art for any DNA sequence determined bythis automated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 95% identical, more typically at least about96% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence may be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide that hybridizes, under stringenthybridization conditions, to a portion of the polynucleotide in anucleic acid molecule of the invention, as described above. By apolynucleotide that hybridizes to a “portion” of a polynucleotide isintended a polynucleotide, either DNA or RNA, hybridizing to at leastabout 15 nucleotides, and more preferably at least about 20 nucleotides,and still more preferably at least about 30 nucleotides, and even morepreferably more than 30 nucleotides of the reference polynucleotide.

For the purpose of the invention, two sequences hybridize when they forma double-stranded complex in a hybridization solution of 6×SSC, 0.5%SDS, 5×Denhardt's solution and 100 μg of non-specific carrier DNA. SeeAusubel et al., supra, at section 2.9, supplement 27 (1994). Sequencesmay hybridize at “moderate stringency,” which is defined as atemperature of 60° C. in a hybridization solution of 6×SSC, 0.5% SDS,5×Denhardt's solution and 100 μg of non-specific carrier DNA. For “highstringency” hybridization, the temperature is increased to 68° C.Following the moderate stringency hybridization reaction, thenucleotides are washed in a solution of 2×SSC plus 0.05% SDS for fivetimes at room temperature, with subsequent washes with 0.1×SSC plus 0.1%SDS at 60° C. for 1 h. For high stringency, the wash temperature isincreased to 68° C. For the purpose of the invention, hybridizednucleotides are those that are detected using 1 ng of a radiolabeledprobe having a specific radioactivity of 10,000 cpm/ng, where thehybridized nucleotides are clearly visible following exposure to X-rayfilm at −70° C. for no more than 72 hours.

The present application is directed to such nucleic acid molecules whichare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a nucleic acid sequence described in of SEQ ID NO:1 or 3. Preferred are nucleic acid molecules which are at least 95%,96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shownin of SEQ ID NO: 1 or 3. Differences between two nucleic acid sequencesmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotidesequence refers to a comparison made between two molecules usingstandard algorithms well known in the art and can be determinedconventionally using publicly available computer programs such as theBLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-3402(1997).

The heterologous sequence utilized in the antisense methods of thepresent invention may be selected so as to produce an RNA productcomplementary to an entire A622 or NBB1 mRNA sequence, or to a portionthereof. The sequence may be complementary to any contiguous sequence ofthe natural messenger RNA, that is, it may be complementary to theendogenous mRNA sequence proximal to the 5′-terminus or capping site,downstream from the capping site, between the capping site and theinitiation codon and may cover all or only a portion of the non-codingregion, may bridge the non-coding and coding region, be complementary toall or part of the coding region, complementary to the 3′-terminus ofthe coding region, or complementary to the 3′-untranslated region of themRNA.

Suitable antisense sequences may be from at least about 13 to about 15nucleotides, at least about 16 to about 21 nucleotides, at least about20 nucleotides, at least about 30 nucleotides, at least about 50nucleotides, at least about 75 nucleotides, at least about 100nucleotides, at least about 125 nucleotides, at least about 150nucleotides, at least about 200 nucleotides, or more. In addition, thesequences may be extended or shortened on the 3′ or 5′ ends thereof.

The particular antisense sequence and the length of the antisensesequence will vary, depending, for example, upon the degree ofinhibition desired and the stability of the antisense sequence.Generally available techniques and the information provided in thisspecification can guide the selection of appropriate A622 or NBB1antisense sequences. With reference to SEQ ID NO: 1 or 3 herein, anoligonucleotide of the invention may be a continuous fragment of A622 orNBB1 cDNA sequence in antisense orientation, of any length that issufficient to achieve the desired effects when transformed into arecipient plant cell.

The present invention may contemplate sense co-suppression of one orboth of A622 and NBB1. Sense polynucleotides employed in carrying outthe present invention are of a length sufficient to suppress, whenexpressed in a plant cell, the native expression of the plant A622 orNBB1 protein in that plant cell. Such sense polynucleotides may beessentially an entire genomic or complementary nucleic acid encoding theA622 or NBB1 enzyme, or a fragment thereof, with such fragmentstypically being at least 15 nucleotides in length. Techniques aregenerally available for ascertaining the length of sense DNA thatresults in suppression of the expression of a native gene in a cell.

In an alternate embodiment of the present invention, plant cells aretransformed with a nucleic acid construct containing a polynucleotidesegment encoding an enzymatic RNA molecule (a “ribozyme”), whichenzymatic RNA molecule is directed against (i.e., cleaves) the mRNAtranscript of DNA encoding A622 or NBB1, as described herein. Ribozymescontain substrate binding domains that bind to accessible regions of thetarget mRNA, and domains that catalyze the cleavage of RNA, preventingtranslation and protein production. The binding domains may compriseantisense sequences complementary to the target mRNA sequence; thecatalytic motif may be a hammerhead motif or other motifs, such as thehairpin motif.

Ribozyme cleavage sites within an RNA target may initially be identifiedby scanning the target molecule for ribozyme cleavage sites (e.g., GUA,GUU or GUC sequences). Once identified, short RNA sequences of 15, 20,30, or more ribonucleotides corresponding to the region of the targetgene containing the cleavage site may be evaluated for predictedstructural features.

The suitability of candidate targets also may be evaluated by testingtheir accessibility to hybridization with complimentaryoligonucleotides, using ribonuclease protection assays as are known inthe art. DNA encoding enzymatic RNA molecules may be produced inaccordance with known techniques. For example, see Cech et al., U.S.Pat. No. 4,987,071; Keene et al., U.S. Pat. No. 5,559,021; Donson etal., U.S. Pat. No. 5,589,367; Torrence et al., U.S. Pat. No. 5,583,032;Joyce, U.S. Pat. No. 5,580,967; Gold et al., U.S. Pat. No. 5,595,877;Wagner et al., U.S. Pat. No. 5,591,601; and U.S. Pat. No. 5,622,854.

Production of such an enzymatic RNA molecule in a plant cell anddisruption of A622 or NBB1 protein production reduces protein activityin plant cells, in essentially the same manner as production of anantisense RNA molecule; that is, by disrupting translation of mRNA inthe cell which produces the enzyme. The term “ribozyme” describes anRNA-containing nucleic acid that functions as an enzyme, such as anendoribonuclease, and may be used interchangeably with “enzymatic RNAmolecule.”

The present invention further includes nucleic acids encoding ribozymes,nucleic acids that encode ribozymes and that have been inserted into anexpression vector, host cells containing such vectors, and methodologyemploying ribozymes to decrease A622 and NBB1 expression in plants.

In one embodiment, the present invention provides double-strandednucleic acid molecules of that mediate RNA interference gene silencing.In another embodiment, the siNA molecules of the invention consist ofduplex nucleic acid molecules containing about 15 to about 30 base pairsbetween oligonucleotides comprising about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, siNA molecules of the inventioncomprise duplex nucleic acid molecules with overhanging ends of about 1to about 32 (e.g., about 1, 2, or 3) nucleotides, for example, about21-nucleotide duplexes with about 19 base pairs and 3′-terminalmononucleotide, dinucleotide, or trinucleotide overhangs. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with blunt ends, where both ends are blunt, or alternatively,where one of the ends is blunt.

An siNA molecule of the present invention may comprise modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

For example, A622 and NBB1 expression may be decreased through geneticengineering methods that are well known in the art. Expression can bereduced by introducing a nucleic acid construct that results inexpression of an RNA comprising a portion of a sequence encoding A622 orNBB1. The portion of the sequence may be in the sense or antisenseorientation. The portion of the sequence may be present in invertedrepeats capable of forming a double-stranded RNA region. Expression maybe reduced by introducing a nucleic acid construct encoding an enzymaticRNA molecule (i.e., a “ribozyme”), which enzymatic RNA molecule isdirected against (i.e., cleaves) the mRNA transcript of DNA encodingA622 or NBB1. Expression may be reduced by introducing a nucleic acidcomprising a portion of an A622 or NBB1 sequence that causes targeted insitu mutagenesis of an endogenous gene, resulting in its inactivation

In one embodiment of the present invention, plant cells are transformedwith a nucleic acid construct containing a mutant allele of one or bothof A622 and NBB that comprises a polynucleotide sequence that suppressesexpression of one or both of A622 and NBB1. Mutant alleles according tothe invention may arise from antisense sequence suppression of one orboth of A622 and NBB1, or sense co-suppression of one or both of A622and NBB1, as described herein. Thus, a mutant allele according to theinvention may comprises an antisense nucleic acid sequence thatexpresses a short interfering RNA that suppresses expression of one orboth of A622 and NBB1 or expression of a gene product encoded by SEQ IDNO: 1 or SEQ ID NO: 3. In an alternative embodiment, the mutant allelemay comprise a sense nucleic acid sequence that suppresses expression ofone or both of A622 and NBB1 or expression of a gene product encoded bySEQ ID NO: 1 or SEQ ID NO: 3. In yet another embodiment, the mutantallele according to the invention comprises a nucleic acid sequence thatencodes a ribozyme which cleaves one or both of A622 and NBB1transcripts. In a preferred embodiment, mutant alleles are at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence described in of SEQ ID NO: 1 or 3. Preferred aremutant alleles which are at least 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid sequence set forth in SEQ ID NO: 1 or 3.Differences between two mutant alleles may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

Sequence Analysis

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch,J. Mol. Biol. 48: 443 (1970); by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet et al., Nucleic Acids Research, 16: 10881-90(1988); Huang et al., Computer Applications in the Biosciences 8: 155-65(1992), and Pearson et al., Methods in Molecular Biology 24: 307-331(1994).

The BLAST family of programs that can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul et al., J.Mol. Biol. 215:403-410 (1990); and, Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix. See Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89:10915 (1998).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5877 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins & Sharp, CABIOS 5:151-153 (1989)) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The following running parameters are preferred for determiningalignments and similarities using BLASTN that contribute to the E valuesand percentage identity for polynucleotide sequences: Unix runningcommand: blastall -p blastn -d embldb -e 10 -G0 -E0 -r 1 -v 30 -b 30 -iqueryseq -o results; the parameters are: -p Program Name [String]; -dDatabase [String]; -e Expectation value (E) [Real]; -G Cost to open agap (zero invokes default behavior) [Integer]; -E Cost to extend a gap(zero invokes default behavior) [Integer]; -r Reward for a nucleotidematch (blastn only) [Integer]; -v Number of one-line descriptions (V)[Integer]; -b Number of alignments to show (B) [Integer]; -i Query File[File In]; and -o BLAST report Output File [File Out] Optional.

The “hits” to one or more database sequences by a queried sequenceproduced by BLASTN, FASTA, BLASTP or a similar algorithm, align andidentify similar portions of sequences. The hits are arranged in orderof the degree of similarity and the length of sequence overlap. Hits toa database sequence generally represent an overlap over only a fractionof the sequence length of the queried sequence.

The BLASTN, FASTA and BLASTP algorithms also produce “Expect” values foralignments. The Expect value (E) indicates the number of hits one can“expect” to see over a certain number of contiguous sequences by chancewhen searching a database of a certain size. The Expect value is used asa significance threshold for determining whether the hit to a database,such as the preferred EMBL database, indicates true similarity. Forexample, an E value of 0.1 assigned to a polynucleotide hit isinterpreted as meaning that in a database of the size of the EMBLdatabase, one might expect to see 0.1 matches over the aligned portionof the sequence with a similar score simply by chance. By thiscriterion, the aligned and matched portions of the polynucleotidesequences then have a probability of 90% of being the same. Forsequences having an E value of 0.01 or less over aligned and matchedportions, the probability of finding a match by chance in the EMBLdatabase is 1% or less using the BLASTN or FASTA algorithm.

According to one embodiment, “variant” polynucleotides, with referenceto each of the polynucleotides of the present invention, preferablycomprise sequences having the same number or fewer nucleic acids thaneach of the polynucleotides of the present invention and producing an Evalue of 0.01 or less when compared to the polynucleotide of the presentinvention. That is, a variant polynucleotide is any sequence that has atleast a 99% probability of being the same as the polynucleotide of thepresent invention, measured as having an E value of 0.01 or less usingthe BLASTN, FASTA, or BLASTP algorithms set at parameters describedabove. Alternatively, variant polynucleotides of the present inventionhybridize to the polynucleotide sequence of SEQ ID NO: 1 or 3, orcomplements, reverse sequences, or reverse complements of thosesequences, under stringent conditions.

The present invention also encompasses polynucleotides that differ fromthe disclosed sequences but that, as a consequence of the degeneracy ofthe genetic code, encode a polypeptide which is the same as that encodedby a polynucleotide of the present invention. Thus, polynucleotidescomprising sequences that differ from the polynucleotide sequencesrecited in SEQ ID NO: 1 or 3 or complements, reverse sequences, orreverse complements thereof, as a result of conservative substitutionsare contemplated by and encompassed within the present invention.Additionally, polynucleotides comprising sequences that differ from thepolynucleotide sequences recited in SEQ ID NO: 1 or 3, or complements,reverse complements or reverse sequences thereof, as a result ofdeletions and/or insertions totaling less than 10% of the total sequencelength are also contemplated by and encompassed within the presentinvention.

In addition to having a specified percentage identity to an inventivepolynucleotide sequence, variant polynucleotides preferably haveadditional structure and/or functional features in common with theinventive polynucleotide. In addition to sharing a high degree ofsimilarity in their primary structure to polynucleotides of the presentinvention, polynucleotides having a specified degree of identity to, orcapable of hybridizing to an inventive polynucleotide preferably have atleast one of the following features: (i) they contain an open readingframe or partial open reading frame encoding a polypeptide havingsubstantially the same functional properties as the polypeptide encodedby the inventive polynucleotide; or (ii) they have domains in common.For example, a variant polynucleotide may encode a polypeptide havingthe ability to suppress A622 or NBB1.

Nucleic Acid Constructs

In accordance with one aspect of the invention, a sequence that reducesnicotinic alkaloid biosynthesis is incorporated into a nucleic acidconstruct that is suitable for plant transformation. For example, such anucleic acid construct can be used to decrease at least one of A622 orNBB1 gene expression in plants. Additionally, an inventive nucleic acidconstruct may decrease one or both of A622 and NBB1 expression, as wellas a polynucleotide sequence encoding a nicotine biosynthesis enzyme.

Accordingly, nucleic acid constructs are provided that comprise asequence that down-regulates nicotinic alkaloid biosynthesis, under thecontrol of a transcriptional initiation region operative in a plant, sothat the construct can generate RNA in a host plant cell.

Recombinant DNA constructs may be made using standard techniques. Forexample, the DNA sequence for transcription may be obtained by treatinga vector containing said sequence with restriction enzymes to cut outthe appropriate segment. The DNA sequence for transcription may also begenerated by annealing and ligating synthetic oligonucleotides or byusing synthetic oligonucleotides in a polymerase chain reaction (PCR) togive suitable restriction sites at each end. The DNA sequence then iscloned into a vector containing suitable regulatory elements, such asupstream promoter and downstream terminator sequences.

Suitable Regulatory Elements

Promoter connotes a region of DNA upstream from the start oftranscription that is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “constitutivepromoter” is one that is active throughout the life of the plant andunder most environmental conditions. Tissue-specific, tissue-preferred,cell type-specific, and inducible promoters constitute the class of“non-constitutive promoters.” “Operably linked” refers to a functionallinkage between a promoter and a second sequence, where the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. In general, “operably linked”means that the nucleic acid sequences being linked are contiguous.

Promoters useful for expression of a nucleic acid sequence introducedinto a cell to reduce expression of A622 or NBB1 may be constitutivepromoters, or tissue-specific, tissue-preferred, cell type-specific, andinducible promoters. Preferred promoters include promoters which areactive in root tissues, such as the tobacco RB7promoter (Hsu et al.Pestic. Sci. 44:9-19 (1995); U.S. Pat. No. 5,459,252) and promoters thatare activated under conditions that result in elevated expression ofenzymes involved in nicotine biosynthesis such as the tobacco RD2promoter (U.S. Pat. No. 5,837,876), PMT promoters (Shoji T. et al.,Plant Cell Physiol, 41:831-839 (2000b); WO 2002/038588) or an A622promoter (Shoji T. et al., Plant Mol Biol, 50:427-440 (2002)).

The vectors of the invention may also contain termination sequences,which are positioned downstream of the nucleic acid molecules of theinvention, such that transcription of mRNA is terminated, and polyAsequences added. Exemplary of such terminators are the cauliflowermosaic virus (CaMV) 35S terminator and the nopaline synthase gene (Tnos)terminator. The expression vector also may contain enhancers, startcodons, splicing signal sequences, and targeting sequences.

Expression vectors of the invention may also contain a selection markerby which transformed plant cells can be identified in culture. Themarker may be associated with the heterologous nucleic acid molecule,i.e., the gene operably linked to a promoter. As used herein, the term“marker” refers to a gene encoding a trait or a phenotype that permitsthe selection of, or the screening for, a plant or plant cell containingthe marker. Usually, the marker gene will encode antibiotic or herbicideresistance. This allows for selection of transformed cells from amongcells that are not transformed or transfected.

Examples of suitable selectable markers include adenosine deaminase,dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidnekinase, xanthine-guanine phospho-ribosyltransferase, glyphosate andglufosinate resistance, and amino-glycoside 3′-O-phosphotranserase(kanamycin, neomycin and G418 resistance). These markers includeresistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.The construct may also contain the selectable marker gene Bar thatconfers resistance to herbicidal phosphinothricin analogs like ammoniumgluphosinate. Thompson et al., EMBO J. 9: 2519-2523 (1987). Othersuitable selection markers are known as well.

Visible markers such as green florescent protein (GFP) may be used.Methods for identifying or selecting transformed plants based on thecontrol of cell division have also been described. See WO 2000/052168and WO 2001/059086.

Replication sequences, of bacterial or viral origin, may also beincluded to allow the vector to be cloned in a bacterial or phage host.Preferably, a broad host range prokaryotic origin of replication isused. A selectable marker for bacteria may be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other DNA sequences encoding additional functions may also be present inthe vector, as is known in the art. For instance, when Agrobacterium isthe host, T-DNA sequences may be included to facilitate the subsequenttransfer to and incorporation into plant chromosomes.

Plants for Genetic Engineering

The present invention comprehends the genetic manipulation of a plant tosuppress nicotinic alkaloid synthesis via introducing a polynucleotidesequence that down-regulates expression of a gene, such as A622 andNBB1, that regulates nicotinic alkaloid synthesis. The result is a planthaving reduced nicotinic alkaloid levels.

In this description, “plant” denotes any nicotinic alkaloid containingplant material that can be genetically manipulated, including but notlimited to differentiated or undifferentiated plant cells, protosomes,whole plants, plant tissues, or plant organs, or any component of aplant such as a leaf, stem, root, bud, tuber, fruit, rhizome, or thelike.

Illustrative plants that can be engineered in accordance with theinvention include but are not limited to tobacco, potato, tomato, eggplant, green pepper, and Atropa belladonna.

Plant Transformation and Selection

Constructs according to the invention may be used to transform any plantcell, using a suitable transformation technique. Both monocotyledonousand dicotyledonous angiosperm or gymnosperm plant cells may betransformed in various ways known to the art. For example, see Klein etal., Biotechnology 4: 583-590 (1993); Bechtold et al., C. R. Acad. Sci.Paris 316:1194-1199 (1993); Bent et al., Mol. Gen. Genet. 204:383-396(1986); Paszowski et al., EMBO J. 3: 2717-2722 (1984); Sagi et al.,Plant Cell Rep. 13: 262-266 (1994). Agrobacterium species such as A.tumefaciens and A. rhizogenes can be used, for example, in accordancewith Nagel et al., Microbiol Lett 67: 325 (1990). Additionally, plantsmay be transformed by Rhizobium, Sinorhizobium or Mesorhizobiumtransformation. Broothaerts et al., Nature 433:629-633 (2005).

For example, Agrobacterium may be transformed with a plant expressionvector via, e.g., electroporation, after which the Agrobacterium isintroduced to plant cells via, e.g., the well known leaf-disk method.Additional methods for accomplishing this include, but are not limitedto, electroporation, particle gun bombardment, calcium phosphateprecipitation, and polyethylene glycol fusion, transfer into germinatingpollen grains, direct transformation (Lorz et al., Mol. Genet. 199:179-182 (1985)), and other methods known to the art. If a selectionmarker, such as kanamycin resistance, is employed, it makes it easier todetermine which cells have been successfully transformed.

The Agrobacterium transformation methods discussed above are known to beuseful for transforming dicots. Additionally, de la Pena et al., Nature325: 274-276 (1987), Rhodes et al., Science 240: 204-207 (1988), andShimamato et al., Nature 328: 274-276 (1989) have transformed cerealmonocots using Agrobacterium. Also see Bechtold et al., C.R. Acad. Sci.Paris 316 (1994), illustrating vacuum infiltration forAgrobacterium-mediated transformation.

For the purposes of this description, a plant or plant cell may betransformed with a plasmid comprising one or more sequences, eachoperably linked to a promoter. For example, an illustrative vector maycomprise a QPT sequence operably linked to a promoter. Likewise, theplasmid may comprise a QPT sequence operably linked to a promoter and anA622 sequence operably linked to a promoter. Alternatively, a plant orplant cell may be transformed with more than one plasmid. For example, aplant or plant cell may be transformed with a first plasmid comprising aQPT sequence operably linked to a promoter, which is distinct from asecond plasmid comprising an A622 or NBB1 sequence. Of course, the firstand second plasmids or portions thereof are introduced into the sameplant cell

Genetically engineered plants of the invention may be produced byconventional breeding. For example, a genetically engineered planthaving down-regulated QPT and A622 activity may be produced by crossinga transgenic plant having reduced QPT expression with a transgenic planthaving reduced A622 expression. Following successive rounds of crossingand selection, a genetically engineered plant having down-regulated QPTand A622 activity can be selected.

The presence of a protein, polypeptide, or nucleic acid molecule in aparticular cell can be measured to determine if, for example, a cell hasbeen successfully transformed or transfected.

Marker genes may be included within pairs of recombination sitesrecognized by specific recombinases such as cre or flp to facilitateremoval of the marker after selection. See U.S. published applicationNo. 2004/0143874.

Transgenic plants without marker genes may be produced using a secondplasmid comprising a nucleic acid encoding the marker, distinct from afirst plasmid that comprises an A622 or NBB1 sequence. The first andsecond plasmids or portions thereof are introduced into the same plantcell, such that the selectable marker gene that is transientlyexpressed, transformed plant cells are identified, and transformedplants are obtained in which the A622 or NBB1 sequence is stablyintegrated into the genome and the selectable marker gene is not stablyintegrated. See U. S. published application No. 2003/0221213. The firstplasmid that comprises an A622 or NBB1 sequence may optionally be abinary vector with a T-DNA region that is completely made up of nucleicacid sequences present in wild-type non-transgenic N. tabacum orsexually compatible Nicotiana species.

Plant cells may be transformed with nucleic acid constructs of thepresent invention without the use of a selectable or visible marker andtransgenic plant tissue and transgenic regenerated plants may beidentified by detecting the presence of the introduced construct by PCRor other methods of detection of specific nucleic acid sequences.Identification of transformed plant cells may be facilitated byrecognition of differences in the growth rate or a morphological featureof said transformed plant cell compared to the growth rate or amorphological feature of a non-transformed plant cell that is culturedunder similar conditions (see WO 2004/076625).

Methods of regenerating a transgenic plant from a transformed cell orculture vary according to the plant species but are based on knownmethodology. For example, methods for regenerating of transgenic tobaccoplants are well-known.

For the purposes of the present description, genetically engineeredplants are selected that have down-regulated expression of at least oneof A622 and NBB1. Additionally, the inventive genetically engineeredplants may have down-regulated expression of a nicotine biosynthesisgene, such as QPT or PMT, and at least one of A622 and NBB1.

Nicotine serves as a natural pesticide which helps protect tobaccoplants from damage by pests and susceptibility of conventionally bred ortransgenic low-nicotine tobacco to insect damage has been reported toincrease. Legg, P. D., et al., Can. J. Cyto., 13:287-291 (1971);Voelckel, C., et al., Chemoecology 11:121-126 (2001); Steppuhn, A., etal., PLoS Biol, 2(8): e217: 1074-1080 (2004). It may therefore bedesirable to additionally transform reduced-nicotine plants produced bythe present methods with a transgene that will confer additional insectprotection, such as gene encoding a Bt insecticidal protein, proteinaseinhibitor, or biotin-binding protein. A transgene conferring additionalinsect protection may be introduced by crossing a transgenicreduced-nicotine plant with a second transgenic plant containing a geneencoding an insect resistance protein.

Quantifying Nicotinic Alkaloid Content

Transgenic plants of the invention are characterized by decreasednicotinic alkaloid content. Decreased nicotinic alkaloid content in thegenetically engineered plant is preferably achieved via decreasedexpression of a nicotine biosynthesis pathway gene, such as A622 orNBB1.

In describing a plant of the invention, the phrase “reduced-nicotine ornicotinic alkaloid content” refers to a quantitative reduction in theamount of nicotinic alkaloid in the plant when compared with anon-transformed control plant. A quantitative decrease in nicotinicalkaloid levels can be assayed by several methods, as for example byquantification based on gas-liquid chromatography, high performanceliquid chromatography, radio-immunoassays, and enzyme-linkedimmunosorbent assays. In the present invention, nicotinic alkaloidlevels were measured by gas-liquid chromatography equipped with acapillary column and an FID detector, as described in Hibi, N. et al.,Plant Physiology 100: 826-835 (1992).

Reduced-Nicotinic-Alkaloid Products

The present invention provides a transgenic plant havingreduced-nicotinic-alkaloid levels. For example, the instant inventioncontemplates reducing nicotine levels by suppressing at least one ofA622 and NBB1 expression. Following selection of a transgenic planthaving suppressed A622 or NBB1 expression and reduced-nicotine content,a variety of products may be made from such a plant.

Because the invention provides a method for reducing alkaloids, TSNAsmay also be reduced because there is a significant, positive correlationbetween alkaloid content in tobacco and TSNA accumulation. For example,a significant correlation coefficient between anatabine and NAT was0.76. Djordjevic et al., J. Agric. Food Chem., 37: 752-756 (1989). TSNAsare a class of carcinogens that are predominantly formed in tobaccoduring curing, processing, and smoking. However, TSNAs are present insmall quantities in growing tobacco plants or fresh cut tobacco. Hecht &Hoffman, J. Natl. Cancer Inst. 58, 1841-4 (1977); Wiernik et al.,RecentAdv. Tob. Sci, 21: 39-80 (1995). Nitrosamines, containing theorganic functional group, N—N═O, are formed from the facile addition ofan N═O group by a nitrosating agent to a nitrogen of a secondary ortertiary amine. This particular class of carcinogens is found only intobacco although they could potentially occur in othernicotine-containing products.

TSNAs are considered to be among the most prominent carcinogens incigarette smoke and their carcinogenic properties are well documented.See Hecht, S. Mutat. Res. 424:127-42 (1999); Hecht, S. Toxicol. 11,559-603 (1998); Hecht, S., et al., Cancer Surv. 8, 273-294 (1989). TSNAshave been cited as causes of oral cancer, esophageal cancer, pancreaticcancer, and lung cancer (Hecht & Hoffman, IARC Sci. Publ. 54-61 (1991)).In particular, TSNAs have been implicated as the causative agent in thedramatic rise of adenocarcinoma associated with cigarette smoking andlung cancer (Hoffmann et al., Crit. Rev. Toxicol. 26, 199-211 (1996)).

The four TSNAs considered to be the most important by levels of exposureand carcinogenic potency and reported to be possibly carcinogenic tohumans are N′-nitrosonornicotine (NNN),4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine(NAT) and N′-nitrosoanabasine (NAB) Reviewed in IARC monographs on theevaluation of the carcinogenic risk of chemical to humans. Lyon (France)Vol 37, pp. 205-208 (1985). These TSNAs are formed by N-nitrosation ofnicotine and of the minor Nicotiana alkaloids that include nornicotine,anatabine, and anabasine.

The following levels of alkaloid compounds have been reported formainstream smoke of non-filter cigarettes (measured in μg/cigarette):nicotine: 100-3000, nornicotine: 5-150, anatabine: 5-15, Anabasine: 5-12(Hoffmann et al., Chem. Res. Toxicol. 14:7:767-790 (2000)). Mainstreamsmoke of U.S. cigarettes, with or without filter tips, contain (measuredin ng/cigarette): 9-180 ng NNK, 50-500 ng NNN, 3-25 ng NAB and 55-300 ngNAT. Hoffmann, et al., J. Toxicol. Environ. Health 41:1-52 (1994). It isimportant to note that the levels of these TSNAs in sidestream smoke are5-10 fold above those in mainstream smoke. Hoffmann, et al (1994).

Xie et al. (2004) reported that Vector 21-41, which is a GEreduced-nicotine tobacco by the down-regulation of QPT, has a totalalkaloid level of about 2300 ppm, which is less than 10 percent of thewild-type tobacco. Mainstream smoke from the Vector 21-41 cigarettes hadless than 10 percent of NNN, NAT, NAB, and NNK compared to such levelsof a standard full flavor cigarette produced from wild-type tobacco.

The strategy for reducing TSNAs by reducing the corresponding tobaccoalkaloid precursors is currently the main focus of agricultural tobaccoresearch. Siminszky et al., Proc. Nat. Acad. Sci. USA 102(41)14919-14924 (2005). Thus, to reduce formation of all TSNAs there is anurgent need to reduce the precursor nicotinic alkaloids as much aspossible by genetic engineering.

Among others, U.S. Pat. Nos. 5,803,081, 6,135,121, 6,805,134, 6,907,887and 6,959,712 and U.S. Published Application Nos. 2005/0034365 and2005/0072047 discuss methods to reduce tobacco-specific nitrosamines(TSNAs).

A reduced-nicotine tobacco product may be in the form of leaf tobacco,shredded tobacco, cut tobacco and tobacco fractions. A reduced-nicotinetobacco product may include cigarette tobacco, cigar tobacco, snuff,chewing tobacco, pipe tobacco, and cigarettes made from GEreduced-nicotine tobacco for use in smoking cessation.

Reduced-nicotine tobacco may also be used to produce reconstitutedtobacco (Recon). Recon is produced from tobacco stems and/or smallerleaf particles by a process that closely resembles typical paper making.This process entails processing the various tobacco portions that are tobe made into Recon and cutting the tobacco into a size and shape thatresembles cut rag tobacco made from whole leaf tobacco. This cut reconthen gets mixed with cut-rag tobacco and is ready for cigarette making.

In addition to traditional tobacco products, such as cigarette and cigartobacco, reduced-nicotine tobacco can be used as source for protein,fiber, ethanol, and animal feeds. See U.S. published application No.2002/0197688. For example, reduced-nicotine tobacco may be used as asource of Rubisco (ribulose bisphosphate carboxylase-oxygenase orfraction 1 protein) because unlike other plants, tobacco-derived Rubiscocan be readily extracted in crystalline form. With the exception ofslightly lower levels of methionine, Rubisco's content of essentialamino acids equals or exceeds that of the FAO Provisional Pattern.Ershoff, B. H., et al. Society for Experimental Biology and Medicine157:626-630 (1978); Wildman, S. G. Photosynthesis Research 73:243-250(2002)).

For biofuels to replace a sizable portion of the world's dependence onnon-renewable energy sources, co-products, such as Rubisco, are requiredto help defray the cost of producing this renewable energy. Greene etal. Growing Energy. How Biofuels Can End America's Oil Dependence;National Resources Defense Counsel (2004). Thus, the greater reductionin nicotinic alkaloids in tobacco, the greater the likelihood of asuccessful tobacco biomass system.

Specific examples are presented below of methods for identifyingsequences encoding enzymes involved in nicotine, as well as forintroducing the target gene to produce plant transformants. They aremeant to be exemplary and not as limitations on the present invention.

Example 1: Preparation of pRNAi-A622 Vector for Reducing AlkaloidContent by Down-Regulating A622 Expression

The plasmid pHANNIBAL, see Wesley et al., Plant J. 27: 581-590 (2001),was modified to produce plasmid pHANNIBAL-X as shown in FIG. 2. A SacIrestriction site between the ampicillin resistance gene (Amp) and 35Spromoter was eliminated by SacI cutting and subsequent DNA blunting andligation. The multi-cloning sites (MCS) were modified as follows. A BamH I restriction site was added to the MCS between the promoter and Pdkintron by inserting an adaptor (5′ TCGAACGGGATCCCGCCGCTCGAGCGG) (SEQ IDNO: 5) between the XhoI and EcoRI sites. A Bam H1 site was eliminatedfrom and a Sac I site was inserted into the MCS between the intron andterminator by inserting an adaptor (5′ GATCAGCTCTAGAGCCGAGCTCGC) (SEQ IDNO: 6) between the BamHI and XbaI sites.

A plant RNAi binary vector was prepared using pHANNIBAL-X using thescheme diagramed in FIG. 3, in which distinct “sense” and “antisense”fragments are first obtained by the addition of specific restrictionsites to the ends of a segment of the gene of interest, and then thesense and antisense fragments are inserted in the desired orientationsin the modified pHANNIBAL-X plasmid.

The DNA segment containing the sense and antisense fragments and theintervening Pdk intron was substituted for the GUS coding region ofpBI121 (Wesley et al., 2001) to produce an RNAi binary vector.

The 814 bp-1160 bp region of the A622 cDNA was used as the dsRNA formingregion (sense chain, antisense chain). PCR was performed using A622 cDNAcloned in pcDNAII as the template and primers with additional basesencoding the indicated restriction enzyme sites, and the target DNAfragment was collected and TA cloned to a pGEM-T vector.

The sequences of the primers used were:

Sense chain A622 F814-XhoI - A622 R1160-KpnI (SEQ ID NOS 7-8)A622 F814-XhoI 5′ CCGCTCGAGCGGTCAGAGGAAGATATTCTCCA 3′ A622 R1160-KpnI 5′GGGGTACCCCTGGAATAAGACGAAAAATAG 3′Antisense chain A622 F814-XbaI - A622 R1160-ClaI (SEQ ID NOS 9-10)A622 F814-XbaI 5′ GCTCTAGAGCTCAGAGGAAGATATTCTCCA 3′ A622 R1160-ClaI 5′CCATCGATGGTGGAATAAGACGAAAAATAG 3′

Recombination with the modified pHANNIBAL-X was performed starting withthe sense chain followed by the antisense chain. The TA cloned DNAfragments were cut with the appropriate restriction enzymes, collected,and ligated to pHANNIBAL-X which was cut with the same restrictionenzymes. The resulting plasmid contains a DNA sequence with invertedrepeats of the A622 fragment separated by the Pdk intron.

The RNAi region was excised from the pHANNIBAL-X with the incorporatedsense and antisense chains by treating with BamH I and Sac I and ligatedto pBI121 from which the GUS coding region had been removed by similartreatment to produce the binary vector pRNAi-A622 for planttransformation, which contains a T-DNA segment (FIG. 4) that contains annptII selectable marker cassette and the A622 RNAi cassette.

Example 2: Suppression of A622 in Tobacco BY-2 Cells

While tobacco BY-2 cell cultures do not normally synthesize nicotinicalkaloids, methyl jasmonate treatment induces expression of genes forknown enzymes in the nicotine biosynthesis pathway and elicits formationof nicotinic alkaloids.

In order to deduce the function of A622, RNAi strain cultured cells wereprepared in which mRNA from pRNAi-A622 was expressed in cultured tobaccoBY-2 cells to suppress A622.

Agrotransformation

The vector (pRNAi-A622) was transformed into Agrobacterium tumefaciensstrain EH105, which was used to transform tobacco BY-2 cells. Themethods for infecting and selecting the tobacco BY-2 cells were asfollows.

Four ml of BY-2 cells which had been cultured for 7 days in 100 ml ofmodified LS medium, see Imanishi et al., Plant Mol. Biol., 38: 1101-1111(1998), were subcultured into 100 ml modified LS medium, and culturedfor 4 days.

One hundred microliters of A. tumefaciens solution, which had beencultured for 1 day in YEB medium, were added to the 4 ml of cells thathad been cultured for 4 days, and the two were cultured together for 40hours in the dark at 27° C.

After culture, the cells were washed twice with modified LS medium toremove the Agrobacteria.

The washed cells were spread on modified LS selection medium containingkanamycin (50 mg/l) and carbenicillin (250 mg/l), and transformed cellswere selected.

After having been cultured for about 2 weeks in the dark at 27° C., thetransformed cells were transferred to a fresh modified LS selectionmedium, and cultured in the dark for 1 week at 27° C.

The transformed cells were then grown in a suspension culture in thedark at 27° C. for 1 week in 30 ml of liquid modified LS medium.

1 ml of the cultured transformed cells was subcultured to 100 ml ofmodified LS medium. The transformed cells were subcultured every 7 daysin the same way as wild-type cells.

Alkaloid Synthesis

10 ml each of transformed BY-2 cells which had been cultured for 7 daysand cultured tobacco cells which had been transformed using a greenfluorescent protein (GFP) expression vector as the control were washedtwice with modified LS medium containing no 2,4-D, and, after additionof modified LS medium containing no 2,4-D to a total of 100 ml, weresuspension cultured at 27° C. for 12 hours.

After addition of 100 μl of methyljasmonate (MeJa) which had beendiluted to 50 μM with DMSO, the cells were suspension cultured for 48hours at 27° C.

Jasmonate treated cells were filtered, collected, and freeze dried.Sulfuric acid, 3 ml of 0.1 N, was added to 50 mg of the freeze-driedsample. The mixture was sonicated for 15 minutes, and filtered. A 28%ammonium solution was added to 1 ml of the filtrate, and centrifuged for10 minutes at 15000 rpm.

One ml of the supernatant was added to an Extrelut-1 column (Merck) andlet sit for 5 minutes. This was eluted with 6 ml of chloroform. Theeluate was then dried under reduced pressure at 37° C. with anevaporator (Taitec Concentrator TC-8).

The dried sample was dissolved in 50 μl of ethanol solution containing0.1% dodecane. A gas chromatograph (GC-14B) equipped with a capillarycolumn and an FID detector was used to analyze the samples. A RESTECRtx-5Amine column (Restec) was used as the capillary column. The columntemperature was maintained at 100° C. for 10 min, elevated to 150° C. at25° C./min, held at 150° C. for 1 min, elevated to 170° C. at 1° C./min,held at 170° C. for 2 min, elevated to 300° C. at 30° C./min, and thenheld at 300° C. for 10 min. Injection and detector temperature was 300°C. One μl of each sample was injected, and nicotinic alkaloids werequantified by the internal standard method.

As shown in FIG. 5, in the transgenic BY-2 lines in which the A622 geneexpression was suppressed by RNAi (A3, A21 A33 and A43 lines), jasmonateelicitation did not result in high accumulation of anatabine (the majoralkaloid in elicited cultured cells), anatalline, nicotine, oranabasine, compared with control cell lines.

RNA Expression

To determine whether the reduction of alkaloid accumulation in theA622-RNAi lines is specifically related to reduction of A622 expression,rather than an indirect effect on the levels of expression of genes forknown enzymes in the nicotine biosynthesis pathway, the levels ofexpression of A622 and other genes was measured in methyl jasmonatetreated lines, transgenic lines, and control lines.

Total RNAs were isolated from wild-type and transgenic BY-2 cell lineswhich were treated with 50 μM MeJA for 48 h. RNA levels of specificgenes were determined by RT-PCR. RNA was extracted using RNeasy Plantmini kit (Qiagen) according to the manufacture's instructions. cDNA wassynthesized using random hexamers and SuperScript First-Strand SynthesisSystem for RT-PCR (Invitrogen). RT-PCR was performed with 5 ng of thecDNA as a template using a TaKaRa ExTaq (Takara Bio) under the followingconditions: for detection of A622, NBB1, AO, QS, QPT, ODC, 22 cycles of94° C. for 30 sec, 57° C. for 30 sec, and 72° C. for 30 sec, fordetection of PMT, 24 cycles of 94° C. for 1 min, 52° C. for 30 sec, and72° C. for 1 min.

A622 primers: (SEQ ID NOS 11-12) A622-07F 5′ ATGGTTGTATCAGAGAAAAGA622-05R 5′ CCTTCTGCCTCTATCATCCTCCTG NBB1 primers: (SEQ ID NOS 13-14)NBB1-01F 5′ATGTTTCCGCTCATAATTCTG NBB1-1365 5′TCTTCGCCCATGGCTTTTCGGTCTAO primers: (SEQ ID NOS 15-16) AO RT-1 5′ CAAAACCAGATCGCTTGGTC AO RT-25′ CACAGCACTTACACCACCTT QS primers: (SEQ ID NOS 17-18) QS RT-1 5′CGGTGGAGCAAAAGTAAGTG QS RT-2 5′ GAAACGGAACAATCAAAGCA QPT primers:(SEQ ID NOS 19-20) QPT RT-1 5′ TCACTGCTACAGTGCATCCT QPT RT-2 5′TTAGAGCTTTGCCGACACCT ODC primers: (SEQ ID NOS 21-22) ODC RT-1 5′CGTCTCATTCCACATCGGTAGC ODC RT-2 5′ GGTGAGTAACAATGGCGGAAGT PMT primers:(SEQ ID NOS 23-24) PMT RT-1 5′ GCCATGATAATGGCAACGAG PMT RT-2 5′TTAGCAGCGAGATAAGGGAA

As shown in FIG. 6, A622 is not induced in A622-silenced lines. Othergenes for known nicotine biosynthetic pathway enzymes are induced. Theseresults provide evidence that A622 is included in the nicotinic alkaloidbiosynthesis pathway, and demonstrate that the nicotinic alkaloidcontent and particularly the nicotine content of plant cells havingnicotine-producing ability can be reduced by down-regulating A622expression.

Example 3: Construction of an Inducible 4622 RNAi Vector

Constitutive suppression of A622 expression in tobacco hairy rootssignificantly inhibited root growth, precluding analysis of nicotinicalkaloids. To circumvent this, an estradiol-inducible gene expressionsystem (XVE system) was developed. The XVE system produces RNAi hairpinmolecules and target genes are suppressed only after addition of aninducer (beta-estradiol) into the culture medium.

The RNAi region containing A622 sense and antisense DNA fragments wasexcised from the pHANNIBAL-X plasmid with Xho I and Xba I, and ligatedinto pBluescript KS which had been digested with Xho I and Xba I. TheRNAi region was then excised with Xho I and Spe I, and was subclonedbetween the XhoI and Spel sites in the MCS of the XVE vector pER8 (ZuoJ. et al, Plant J., 24: 265-273 (2000)) to produce the binary vectorpXVE-A622RNAi.

The T-DNA region of pXVE-A622RNAi (See FIG. 7) contains a cassette forestradiol-inducible expression of the chimeric transcription factor XVE,an hpt selectable marker cassette, and a cassette in which expression ofthe A622 RNAi is under the control of the LexA-46 promoter, which isactivated by XVE.

Example 4: Suppression of A622 in Tobacco Hairy Roots

The binary vector pXVE-A622RNAi was introduced to Agrobacteriumrhizogenes strain 15834 by electroporation. N. tabacum cv. Petit HavanaSR1 plants were transformed by A. rhizogenes using a leaf-disc method,as described by Kanegae et al., Plant Physiol. 105(2):483-90. (1994).Hygromycin resistance (15 mg/L in B5 medium) was used to selecttransformed roots. Transgenic hairy roots were grown at 27° C. in thedark.

Transgenic hairy roots carrying the T-DNA from pXVE-A622RNAi were grownin the B5 medium for 10 days and then gene silencing was induced byaddition of 17-beta-estradiol (2 μM) for 4 days. RT-PCR analysis showedthat A622 expression was efficiently suppressed in tobacco hairy rootlines A8 and A10 transformed with the estradiol-inducible A622suppression construct after the estradiol addition. See FIG. 8A.

Total RNA was extracted from hairy roots by using RNeasy Plant mini kit(Qiagen). cDNA was synthesized by using random hexamers and SuperScriptFirst-Strand Synthesis System for RT-PCR (Invitrogen). RT-PCR wascarried with 2.5 ng of the cDNA as a template using a TaKaRa ExTaq(Takara Bio) under the following conditions: for detection of A622, 22cycles of 94° C. for 30 sec, 57° C. for 30 sec, and 72° C. for 30 sec;for detection of α-tubulin, 24 cycles of 94° C. for 1 min, 52° C. for 30sec, and 72° C. for 1 min.

Primers for A622 detection; (SEQ ID NOS 11-12) A622-07F 5′ATGGTTGTATCAGAGAAAAG A622-05R 5′ CCTTCTGCCTCTATCATCCTCCTGPrimers for αc-tubulin detection; (SEQ ID NOS 25-26) Tub RT-1 5′AGTTGGAGGAGGTGATGATG Tub RT-2 5′ TATGTGGGTCGCTCAATGTC

Nicotine contents were measured in hairy root lines transformed with aninducible A622 suppression construct without (−) and after induction ofsuppression with estradiol (+). The RT-PCR graph in FIG. 8B shows thatA622 expression was already partially suppressed before estradiolinduction. This is especially true with the #8 line. Nicotine contentsvaried among the A622 suppressed lines but were lower in the A622suppressed lines than in the wild-type hairy roots.

Example 5: Identification of NBB1 as a Gene Regulated by the NIC Loci

A cDNA micro-array prepared from a Nicotiana sylvestris-derived cDNAlibrary (Katoh et al., Proc. Japan Acad., Vol. 79, Ser. B, No. 6, pp.151-154 (2003)) was used to search for novel genes which are controlledby the nicotine biosynthesis regulatory NIC loci.

N. sylvestris cDNAs were amplified by PCR and spotted onto mirror-coatedslides (type 7 star, Amersham) by using Amersham Lucidea array spotter.DNA was immobilized on the slide surface by UV crosslinking (120 mJ/m²).N. tabacum Burley 21 plantlets (WT and nic1nic2) were grown onhalf-strength B5 medium supplemented with 1.5% (W/V) sucrose and 0.35%(W/V) gellan gum (Wako) in Agripot containers (Kirin).

Roots of eight-week-old plantlets were harvested, immediately frozenwith liquid nitrogen, and kept at −80° C. until use. Total RNA wasisolated using Plant RNeasy Mini kit (Qiagen) from the frozen roots, andmRNA was purified using GenElute mRNA Miniprep kit (Sigma). cDNA wassynthesized from 0.4 g of the purified mRNA by using LabelStar Array Kit(Qiagen) in the presence of Cy3 or Cy5-dCTP (Amersham). cDNAhybridization to the microarray slides and post-hybridization washeswere performed using a Lucida Pro hybrid-machine (Amersham). Microarrayswere scanned using an FLA-8000 scanner (Fujifilm). Acquired array imageswere quantified for signal intensity with ArrayGauge software(Fujifilm). cDNA probes from wild type and nic1nic2 tobacco were labeledwith Cy3 and Cy5 in reciprocal pair-wise combinations. Hybridizationsignals were normalized by accounting for the total signal intensity ofdyes. cDNA clones which hybridized to wild-type probes more than twiceas strongly compared to nic1nic2 probes were identified, and theseincluded NBB1.

Full-length NBB1 cDNA was obtained by 5′- and 3′-RACE from total RNA ofN. tabacum by using a SMART RACE cDNA Amplification Kit (Clontech).

The nucleotide sequence of the NBB1 cDNA insert was determined on bothstrands using an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems)and a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).The nucleotide sequence of NBB1 is set forth in SEQ ID NO: 3. The aminoacid sequence encoded by the nucleotide sequence is set forth in SEQ IDNO: 4. The protein sequence includes a FAD-binding motif. A putativevacuolar signal peptide is located at N-terminus.

Example 6: Characterization of NBB1

NBB1 expression was investigated in tobacco plants by Northern blotanalysis.

RNA was extracted from plant bodies which had been treated withmethyljasmonate vapor, using Nicotiana tabacum cv. Burley 21(abbreviated below as WT) and mutants nic1, nic2 and nic1 nic2 havingmutations introduced in the Burley 21 background. Cultivation was in asterile sealed environment, and the plants were raised for 2 months at25° C. with 150 t mole photons/m² of light (16 h light, 8 h dark) on½×B5 medium (3% sucrose, 0.3% gellan gum). Methyl jasmonate treatmentwas accomplished by adding 0.5 mL of 100 μM methyl jasmonate to anAgripot container (Kirin, Tokyo) with a solid medium capacity of 80 cm³and a gas capacity of 250 cm³ containing the plants. The treatment timeswere set at 0 h and 24 h. The root parts and leaf parts (2^(nd) through6^(th) leaves from a plant body with a total of 7 to 10 leaves) werecollected from the plant bodies and immediately stored frozen usingliquid nitrogen.

RNA was extracted using an RNeasy Midi Kit (Qiagen) according to themanufacturer's protocol. However, polyvinyl pyrrolidine was added to aconcentration of 1% to the RLT. The column operation was performed twiceto increase the purity of the RNA.

RNA blotting was carried out according to the ordinary methods given bySambrook and Russell (Sambrook, J. et al., Molecular Cloning, ColdSpring Harbor Laboratory, Chapter 7 (2001)).

The sequence fragment from 1278 bp through the end (1759 bp) of the NBB1nucleotide sequence (SEQ ID NO: 3) was used as the probe template. Thetemplate was prepared by amplification from the cDNA clone using PCRusing the following primers:

(SEQ ID NOS 27-28) primer 1: GGAAAACTAACAACGGAATCTCTprimer 2: GATCAAGCTATTGCTTTCCCT

The probe was labeled with ³²P using a Bcabest labeling kit (Takara)according to the manufacturer's instructions. Hybridization wasaccomplished using ULTRAhyb (Ambion) as the buffer according to themanufacturer's protocol.

PMT probe was prepared from a PMT sequence cloned into a pcDNAII vectorin E. coli (Hibi et al., 1994). The plasmid was extracted and purifiedfrom the E. coli using a QIAprep Spin Miniprep Kit (Qiagen), treatedwith the restriction enzymes XbaI and HindIII by ordinary methods, andrun through agarose gel electrophoresis, and about 1.5 kb DNA fragmentswere collected. A QIAquick Gel Extraction Kit (Qiagen) was used forcollection. The collected DNA fragments were ³²P labeled by the samemethods used for the NBB1 probe, and hybridized. The results are shownin FIG. 9.

As FIG. 9 clearly shows, NBB1 and PMT have the same pattern ofexpression in tobacco plants. Evidence that NBB1 is involved in nicotinebiosynthesis is that, like PMT and A622, NBB1 is under the control ofthe NIC genes, and it exhibits a similar pattern of expression to PMTand A622.

Example 7: Phylogenetic Analysis of NBB1

NBB1 polypeptide has 25% identity and 60% homology to the Eschscholziacalifornica berberine bridge enzyme (BBE). (Dittrich H. et al., Proc.Natl. Acad. Sci. USA, Vol. 88, 9969-9973 (1991)). An alignment of theNBB1 polypeptide with EcBBE is shown in FIG. 10.

A phylogenetic tree was constructed using the sequences of NBB1polypeptide and plant BBE-like polypeptides (based on Carter andThornburg, Plant Physiol. 134, 460-469 (2004). The phylogenetic analysiswas performed using neighbor-joining method with the CLUSTAL W program.Numbers indicate bootstrap values from 1,000 replicates. The sequencesused were: EcBBE, California poppy BBE (GenBank accession no. AF005655);PsBBE, opium poppy (Papaver somniferum) probable reticuline oxidase(AF025430); BsBBE, barberry (Berberis stolonifera) BBE (AF049347);VuCPRD2, cowpea (Vigna unguiculata) drought-induced protein (AB056448);NspNEC5, Nicotiana sp. Nectarin V (AF503441/AF503442); HaCHOX, sunflower(Helianthus annuus) carbohydrate oxidase (AF472609); LsCHOX, lettuce(Lactuca sativa) carbohydrate oxidase (AF472608); and 27 Arabidopsisgenes (At1g01980, Atg11770, At1g26380, At1g26390, At1g26400, At1g26410,At1g26420, At1g30700, At1g30710, At1g30720, At1g30730, At1g30740,At1g30760, At1g34575, At2g34790, At2g34810, At4g20800, At4g20820,At4g20830, At4g20840, At4g20860, At5g44360, At5g44380, At5g44390,At5g44400, At5g44410, and At5g44440).

The results are shown in FIG. 11. The three known BBEs form a separateclade and are underlined and indicated as “True BBEs.” The sequence ofNBB1 is not highly similar to any of the BBE or BBE-like proteins, andis separated from the other sequences at the base of the tree. The onlyother BBE like protein described from the genus Nicotiana, nectarin V, aprotein described in nectar of the a hybrid ornamental Nicotianalangsdorffii×N. sanderae, Carter and Thornburg (2004), clusters with thecowpea drought-induced protein and several putative BBE-like proteinsfrom Arabidopsis. Because the nectar of the ornamental tobacco lacksalkaloids and nectarin V has glucose oxidase activity, it was concludedthat nectarin V is involved in antimicrobial defense in flowers and isnot likely to have any role in alkaloid synthesis. Id.

Example 8: Preparation of NBB1 Suppression Construct

The 342-bp DNA fragment of the NBB1 cDNA was amplified by PCR and clonedinto pGEM-T vector using the following primers.

Antisense chain (SEQ ID NOS 29-30) NBB1-20E-EcoRI 5′CCGGAATTCGCACAGTGGAATGAAGAGGACG 3′ NBB1-18R-XhoI 5′CCGCTCGAGGCGTTGAACCAAGCATAGGAGG 3′ Sense chain (SEQ ID NOS 31-32)NBB1-16F-ClaI 5′ CCATCGATGCACAGTGGAATGAAGAGGACG 3′ NBB1-19R-XbaI 5′GCTCTAGAGCGTTGAACCAAGCATAGGAGG 3′

Resultant PCR products were digested with EcoRI and XhoI for theantisense insertion, and with ClaI and XbaI for sense chain insertion.The sense DNA fragment was subcloned into the pHANNIBAL-X, followed byinsertion of the antisense fragment. The resulting plasmid contained ainverted repeat of the NBB1 fragment, separated by the Pdk intron.

The RNAi region was excised from the pHANNIBAL-X with BamH I and Sac I,and ligated into pBI121 to replace the GUS coding region and to producethe binary vector pRNAi-NBB1. The T-DNA region of pHANNIBAL-NBB1 3′ (SeeFIG. 12) contains an nptII selectable marker cassette and cassette forexpression of a hairpin RNAi with a double-stranded region correspondingto a 3′ fragment of NBB1.

Example 9: NBB1 Suppression in Tobacco BY-2 Cells

Methyl jasmonate treatment of Tobacco BY-2 cells induces NBB1 expressionin addition to expression of genes for known enzymes in the nicotinebiosynthesis pathway. The effects of NBB1 suppression was tested in BY-2cells.

The vector pRNAi-NBB1 was introduced into A. tumefaciens strain EHA105,which was used to transform tobacco BY-2 cells. BY-2 cells were culturedin 100 ml of modified LS medium. Agrobacterium tumefaciens cells (100μl) in YEB medium were added to 4 ml of BY-2 cells and cultured for 40hours in the dark at 27° C. After infected tobacco cells were washedtwice with modified LS medium, washed tobacco cells were spread onmodified LS agar medium containing kanamycin (50 mg/l) and carbenicillin(250 mg/l). After 2 weeks in the dark at 27° C., growing tobaccocalluses were transferred to fresh LS selection medium with the sameantibiotics, and cultured in the dark at 27° C. for one more week.Growing tobacco cells were transferred to liquid modified LS mediumwithout antibiotics. The transformed tobacco cells were subcultured at a7-day intervals.

Cultured tobacco cells were cultured with modified LS medium without2,4-D, at 27° C. for 12 hours. After 100 μl of methyljasmonate (MeJA)dissolved in DMSO was added to 100 mL of the tobacco suspension cultureto give a final concentration of 50 μM, tobacco cells were cultured foran additional 48 hours. MeJA-treated cells were filtered, collected, andfreeze dried. Sulfuric acid, 3 ml of 0.1 N, was added to 100 mg of thefreeze-dried sample. The mixture was sonicated for 15 minutes, andfiltered. A 28% ammonium solution was added to 1 ml of the filtrate, andcentrifuged for 10 minutes at 15000 rpm. One ml of the supernatant wasadded to an Extrelut-1 column (Merck) and eluted with 6 ml ofchloroform. The eluate was then dried under reduced pressure at 37° C.with an evaporator (Taitec Concentrator TC-8). The dried sample wasdissolved in 50 μl of ethanol solution containing 0.1% dodecane. A gaschromatograph (GC-14B) equipped with a capillary column and an FIDdetector was used to analyze the samples. A RESTEC Rtx-5Amine column(Restec) was used as the capillary column. The column temperature wasmaintained at 100° C. for 10 min, elevated to 150° C. at 25° C./min,held at 150° C. for 1 min, elevated to 170° C. at 1° C./min, held at170° C. for 2 min, elevated to 300° C. at 30° C./min, and then held at300° C. for 10 min. Injection and detector temperature was 300° C. Oneμl of each sample was injected, and nicotinic alkaloids were quantifiedby the internal standard method.

Accumulation of nicotinic alkaloids following methyl jasmonateelicitation was greatly reduced in NBB1-suppressed BY-2 cell lines (N37and N40) compared with wild-type tobacco cells (See FIG. 5).

To determine whether the reduction of alkaloid accumulation in theNBB1-RNAi lines is specifically related to reduction of NBB1 expression,rather than an indirect effect on the levels of expression of genes forknown enzymes in the nicotine biosynthesis pathway, the levels ofexpression of NBB1 and other genes was measured in methyl jasmonatetreated lines transgenic and control lines.

Total RNAs were isolated from wild-type and transgenic BY-2 cell lineswhich had been treated with 50 μM MeJA for 48 h. RNA levels of specificgenes were determined by RT-PCR. The results are shown in FIG. 6.

In NBB1-silenced lines induction of NBB1 is not observed, but inductionof known genes of the nicotine biosynthetic pathway still occurs, aswell as induction of A622. Note also that induction of NBB1 is notaffected in A662-suppressed lines.

These results demonstrate that NBB1 reductase is included in thenicotinic alkaloid biosynthesis pathway, and that the nicotinic alkaloidcontent, and particularly, the nicotine content of plant cells havingnicotine-producing ability can be decreased by down-regulating NBB1expression.

Example 10: NBB1 Suppression in Tobacco Hairy Roots

Tobacco SR-1 hairy roots accumulate nicotine as the major alkaloid. Theeffect of NBB1 suppression on alkaloid accumulation in hairy roots wasstudied.

The binary vector pRNAi-NBB1 3′ was introduced into A. rhizogenes strain15834 by electroporation. N. tabacum cv. Petit Havana SR1 plants weretransformed by A. rhizogenes using a leaf-disc method, as describedabove for suppression of A622 in tobacco hairy roots. Hairy roots wereselected and cultured and alkaloids were extracted, purified, andanalyzed as described above.

When NBB1 expression was suppressed by RNAi, transgenic root lines (HN6,HN19, HN20 and HN29) contained highly reduced levels of nicotinecompared with the control cell line, as well as reduced levels ofanatabine (See FIG. 13).

Transgenic hairy roots carrying pHANNIBAL-NBB1 3′ were grown in B5medium for two weeks, and gene expression was analyzed by RT-PCR. NBB1expression was specifically suppressed in four transgenic lines (SeeFIG. 14). NBB1 Expression of other genes for enzymes in the nicotinebiosynthetic pathway was not affected.

The results demonstrate that reduced-nicotine accumulation results fromreduced NBB1 expression, not a lack of expression of genes for knownenzymes of the nicotine biosynthesis pathway.

Example 11: NBB1 Suppression in Transgenic Tobacco Plants

Two attB-NBB1 fragments were amplified by PCR from the NBB1 cDNA inpGEM-T vector using a primer set of NBB1-aatB 1 and attB1 adapter, and aset of NBB1-attB2 and attB2 adapter.

Gene-specific primers: (SEQ ID NOS 33-34) NBB1-attB1 5′AAAAAGCAGGCTTCGAAGGAGATAGAACCATGGTTCCGCTCATAATT CTGATCAGCTT NBB1-attB25′ AGAAAGCTGGGTCTTCACTGCTATACTTGTGCTCTTGA Adapter primers:(SEQ ID NOS 35-36) attB1 adapter 5′ GGGGACAAGTTTGTACAAAAAAGCAGGCTattB2 adapter 5′ GGGGACCACTTTGTACAAGAAAGCTGGGT

The PCR conditions used were those recommended by the manufacture. Anentry clone pDONR221-NBB1-1 was created by BP recombination reactionsbetween the attB-NBB1 PCR products and pDONR221 (Invitrogen).

The NBB1 ORF was transferred from the pDONR221-NBB1-1 vector to aGATEWAY binary vector pANDA 35HK which was designed to express a dsRNAwith GUS partial fragment under the CaMV35S promoter (Dr. Ko Shimamoto,NAIST) by LR reaction. The resultant NBB1 RNAi vector is referred to aspANDA-NBB1full.

The T-DNA of pANDA-NBB1full (See FIG. 15) contains an nptII selectablemarker cassette, an NBB1 RNAi cassette in which the full length codingregion of NBB1 is present in inverted repeats separated by a GUS linker,and an hpt selectable marker cassette.

The binary vector pANDA-NBB1 full was introduced to A. tumefaciensstrain EHA105 by electroporation. N. tabacum cv. Petit Havana SR1 plantswere transformed by A. tumefaciens using a leaf-disk method, basicallyas described by Kanegae et al., Plant Physiol. 105, 483-490 (1994).Hygromycin resistance (30 mg/L in MS medium) was used for selection.Transgenic plants were regenerated from the leaf discs as described andgrown at 27° C. under continuous light in a growth chamber.

Leaf tissue was collected from TO generation plants grown for 36 days.Alkaloids were extracted, purified, and analyzed as described above.Levels of nicotine in the leaves of plants from lines transformed withthe NBB1 suppression vector pANDA-NBB1full were reduced compared to wildtype (See FIG. 16). Leaves of transgenic lines (#6, #14 and #22)contained levels of nicotine only about 16% of the level in leaves ofwild type plants.

Sequence Listing SEQ ID NO: 1 (A622 polynucleotide)AAAAATCCGATTTAATTCCTAGTTTCTAGCCCCTCCACCTTAACCCGAAGCTACTTTTTTTCTTCCCCTAGGAGTAAAATGGTTGTATCAGAGAAAAGCAAGATCTTAATAATTGGAGGCACAGGCTACATAGGAAAATACTTGGTGGAGACAAGTGCAAAATCTGGGCATCCAACTTTCGCTCTTATCAGAGAAAGCACACTCAAAAACCCCGAGAAATCAAAACTCATCGACACATTCAAGAGTTATGGGGTTACGCTACTTTTTGGAGATATATCCAATCAAGAGAGCTTACTCAAGGCAATCAAGCAAGTTGATGTGGTGATTTCCACTGTCGGAGGACAGCAATTTACTGATCAAGTGAACATCATCAAAGCAATTAAAGAAGCTGGAAATATCAAGAGATTTCTTCCTTCAGAATTTGGATTTGATGTGGATCATGCTCGTGCAATTGAACCAGCTGCATCACTCTTCGCTCTAAAGGTAAGAATCAGGAGGATGATAGAGGCAGAAGGAATTCCATACACATATGTAATCTGCAATTGGTTTGCAGATTTCTTCTTGCCCAACTTGGGGCAGTTAGAGGCCAAAACCCCTCCTAGAGACAAAGTTGTCATTTTTGGCGATGGAAATCCCAAAGCAATATATGTGAAGGAAGAAGACATAGCGACATACACTATCGAAGCAGTAGATGATCCACGGACATTGAATAAGACTCTTCACATGAGACCACCTGCCAATATTCTATCCTTCAACGAGATAGTGTCCTTGTGGGAGGACAAAATTGGGAAGACCCTCGAGAAGTTATATCTATCAGAGGAAGATATTCTCCAGATTGTACAAGAGGGACCTCTGCCATTAAGGACTAATTTGGCCATATGCCATTCAGTTTTTGTTAATGGAGATTCTGCAAACTTTGAGGTTCAGCCTCCTACAGGTGTCGAAGCCACTGAGCTATATCCAAAAGTGAAATACACAACCGTCGACGAGTTCTACAACAAATTTGTCTAGTTTGTCGATATCAATCTGCGGTGACTCTATCAAACTTGTTGTTTCTATGAATCTATTGAGTGTAATTGCAATAATTTTCGCTTCAGTGCTTTTGCAACTGAAATGTACTAGCTAGTTGAACGCTAGCTAAATTCTTTACTGTTGTTTTCTATTTTTCGTCTTATTCCA SEQ ID NO: 2 (A622 polypeptide)MVVSEKSKILIIGGTGYIGKYLVETSAKSGHPTFALIRESTLKNPEKSKLIDTFKSYGVTLLFGDISNQESLLKAIKQVDVVISTVGGQQFTDQVNIIKAIKEAGNIKRFLPSEFGFDVDHARAIEPAASLFALKVRIRRMIEAEGIPYTYVICNWFADFFLPNLGQLEAKTPPRDKVVIFGDGNPKAIYVKEEDIATYTIEAVDDPRTLNKTLHMRPPANILSFNEIVSLWEDKIGKTLEKLYLSEEDILQIVQEGPLPLRTNLAICHSVFVNGDSANFEVQPPTGVEATELYPKVKYT TVDEFYNKFVSEQ ID NO: 3 (NBB1 polynucleotide)ACGCGGGGAGAAATACATACAACATGTTTCCGCTCATAATTCTGATCAGCTTTTCACTTGCTTCCTTGTCTGAAACTGCTACTGGAGCTGTTACAAATCTTTCAGCCTGCTTAATCAACCACAATGTCCATAACTTCTCTATTTACCCCACAAGTAGAAATTACTTTAACTTGCTCCACTTCTCCCTTCAAAATCTTCGCTTTGCTGCACCTTTCATGCCGAAACCAACCTTCATTATCCTACCAAGCAGTAAGGAGGAGCTCGTGAGCACCATTTTTTGTTGCAGAAAAGCATCTTATGAAATCAGAGTAAGGTGCGGCGGACACAGTTACGAAGGAACTTCTTACGTTTCCTTTGACGCTTCTCCATTCGTGATCGTTGACTTGATGAAATTAGACGACGTTTCAGTAGATTTGGATTCTGAAACAGCTTGGGCTCAGGGCGGCGCAACAATTGGCCAAATTTATTATGCCATTGCCAAGGTAAGTGACGTTCATGCATTTTCAGCAGGTTCGGGACCAACAGTAGGATCTGGAGGTCATATTTCAGGTGGTGGATTTGGACTTTTATCTAGAAAATTCGGACTTGCTGCTGATAATGTCGTTGATGCTCTTCTTATTGATGCTGATGGACGGTTATTAGACCGAAAAGCCATGGGCGAAGACGTGTTTTGGGCAATCAGAGGTGGCGGCGGTGGAAATTGGGGCATTGTTTATGCCTGGAAAATTCGATTACTCAAAGTGCCTAAAATCGTAACAACTTGTATGATCTATAGGCCTGGATCCAAACAATACGTGGCTCAAATACTTGAGAAATGGCAAATAGTTACTCCAAATTTGGTCGATGATTTTACTCTAGGAGTACTGCTGAGACCTGCAGATCTACCCGCGGATATGAAATATGGTAATACTACTCCTATTGAAATATTTCCCCAATTCAATGCACTTTATTTGGGTCCAAAAACTGAAGTTCTTTCCATATCGAATGAGACATTTCCGGAGCTAGGCGTTAAGAATGATGAGTGCAAGGAAATGACTTGGGTAGAGTCAGCACTTTTCTTCTCCGAATTAGCTGACGTTAACGGGAACTCGACTGGTGATATCTCCCGTCTGAAAGAACGTTACATGGACGGAAAAGGTTTTTTCAAAGGCAAAACGGACTACGTGAAGAAGCCAGTTTCAATGGATGGGATGCTAACATTTCTTGTGGAACTCGAGAAAAACCCGAAGGGATATCTTGTCTTTGATCCTTATGGCGGAGCCATGGACAAGATTAGTGATCAAGCTATTGCTTTCCCTCATAGAAAAGGTAACCTTTTCGCGATTCAGTATCTAGCACAGTGGAATGAAGAGGACGATTACATGAGCGACGTTTACATGGAGTGGATAAGAGGATTTTACAATACAATGACGCCCTTTGTTTCAAGCTCGCCAAGGGGAGCTTATATCAACTACTTGGATATGGATCTTGGAGTGAATATGGTCGACGACTACTTATTGCGAAATGCTAGTAGCAGTAGTCCTTCTTCCTCTGTTGATGCTGTGGAGAGAGCTAGAGCGTGGGGTGAGATGTATTTCTTGCATAACTATGATAGGTTGGTTAAAGCTAAGACACAAATTGATCCACTAAATGTTTTTCGACATGAACAGAGTATTCCTCCTATGCTTGGTTCAACGCAAGAGCACAAGTATAGCAGTGAATGAGATTTAAAATGTACTACCTTGAGAGAGATTCCGTTGTTAGTTTTCC SEQ ID NO: 4(NBB1 polypeptide) MFPLIILISFSLASLSETATGAVTNLSACLINHNVHNFSIYPTSRNYFNLLHFSLQNLRFAAPFMPKPTFIILPSSKEELVSTIFCCRKASYEIRVRCGGHSYEGTSYVSFDASPFVIVDLMKLDDVSVDLDSETAWAQGGATIGQIYYAIAKVSDVHAFSAGSGPTVGSGGHISGGGFGLLSRKFGLAADNVVDALLIDADGRLLDRKAMGEDVFWAIRGGGGGNWGIVYAWKIRLLKVPKIVTTCMIYRPGSKQYVAQILEKWQIVTPNLVDDFTLGVLLRPADLPADMKYGNTTPIEIFPQFNALYLGPKTEVLSISNETFPELGVKNDECKEMTWVESALFFSELADVNGNSTGDISRLKERYMDGKGFFKGKTDYVKKPVSMDGMLTFLVELEKNPKGYLVFDPYGGAMDKISDQATAFPHRKGNLFAIQYLAQWNEEDDYMSDVYMEWIRGFYNTMTPFVSSSPRGAYINYLDMDLGVNMVDDYLLRNASSSSPSSSVDAVERARAWGEMYFLHNYDRLVKAKTQIDPLNVFRHEQSIPPMLGS TQEHKYSSE

1-27. (canceled)
 28. A primer or probe pair capable of amplifying ordetecting a nucleic acid molecule having at least about 80% identity toSEQ ID NO:
 3. 29. The primer or probe pair of claim 28, wherein theprobe pair comprises isolated nucleic acid molecules comprising: (a) afirst nucleic acid molecule comprising contiguous nucleotides of SEQ IDNO: 3; and (b) and a second nucleic acid molecule comprising contiguousnucleotides of SEQ ID NO: 3, wherein the first and second nucleic acidmolecules function together as primers or probes for a nucleic acidsequence having at least about 80% identity to SEQ ID NO:
 3. 30. Amethod for identifying a tobacco plant comprising a mutant allele ofNBB1, comprising: (a) obtaining a nucleic acid sample from one or moretobacco plants; and (b) contacting the sample with a nucleic acid primercomprising at least about 15 contiguous nucleotides of SEQ ID NO: 3 orits complement; and (c) identifying a sample having a nucleic acidsequence that is at least about 80% identical to SEQ ID NO:
 3. 31. Themethod of claim 30, wherein the identifying method step comprisesdetecting formation of a heteroduplex, wherein the heteroduplexcomprises a nucleic acid sequence that is at least about 80% identicalto SEQ ID NO:
 3. 32. A method for identifying a tobacco plant withreduced NBB1 expression, comprising: (a) measuring NBB1 expression in apopulation of tobacco plants; and (b) selecting a tobacco plant withreduced NBB1 expression relative to wild-type NBB1 levels.
 33. Themethod of claim 32, wherein the measuring of NBB1 expression uses aprimer or probe pair capable of amplifying or detecting a nucleic acidmolecule having at least about 80% identity to SEQ ID NO:
 3. 34. Atobacco plant identified with the method of claim 32, wherein the planthas reduced NBB1 expression.
 35. A product produced from the tobaccoplant of claim
 34. 36. The product of claim 35, wherein the product isselected from the group consisting of smoking cessation products,cigarettes, cigarette tobacco, cigars, cigar tobacco, snus, pipe tobaccoand chewing tobacco.
 37. The product of claim 35, wherein the product isselected from the group consisting of a food product, food ingredient,feed product, feed ingredient, nutritional supplement, and biofuels.